Predictive biomarkers for ctla-4 blockade therapy and for pd-1 blockade therapy

ABSTRACT

Biomarkers are described for predicting the efficacy, risk of relapse, risk of an immune related adverse event (irAE), or combination thereof for a CTLA-4 blockade treatment, such as ipilimumab, in a subject with melanoma. Biomarkers are also described for predicting the efficacy and clinical benefit for a PD-1 blockade treatment, such as a PD-1 blocking antibody, in a subject with melanoma.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.61/644,004, filed May 8, 2012, and U.S. Provisional Application No.61/644,988, filed May 9, 2012. Application No. 61/644,004, filed May 8,2012, and Application No. 61/644,988, filed May 9, 2012, are herebyincorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government Support under Grant No. R01 CA109307 and Grant No. CA 129594 awarded by the National Institutes ofHealth (NIH). The Government has certain rights in the invention.

FIELD OF THE INVENTION

The invention is generally related to the field of predictivebiomarkers, more particularly to biomarkers predictive for CTLA-4blockade treatment and to biomarkers predictive for PD-1 blockadetreatment.

BACKGROUND OF THE INVENTION

T cells play a pivotal role in anti-tumor responses, in the developmentof immune tolerance to self, and in autoimmunity. Cytotoxic TLymphocyte-Associated antigen 4 (CTLA-4) is a surface receptor on Tlymphocytes that down-regulates pathways of T-cell activation (Melero I,et al. Nat Rev Cancer. 7:95-106 (2007)), serving as an immune checkpoint molecule. It is expressed intracellularly in resting T cells,transported to the T cell surface after activation of the T cellreceptor (TCR). TCR engagement leads to tyrosine phosphorylation ofCTLA-4 via the SRC kinase and releases it from AP50, resulting in itssurface expression within 48 hours of T cell activation, leading to Tcell tolerance and ancrgy. CTLA-4 expression is associated withdecreased proliferation with cell cycle arrest at the G₁-S interface anddiminished cytokine secretion (Chambers C A, et al. Annu Rev Immunol.19:565-94 (2001); Greenwald R J, et al. Immunity. 14:145-55 (2001)). Itdecreases cell proliferation through the inhibition of mitogen-activated(MAP) kinases but promotes T cell survival through the binding ofphosphoinositol-3 kinase and activating protein kinase B (PKB/AKT)resulting in T cell ancrgy and tolerance without T cell death (SchnciderH, ct al. PLoS One. 3:e3842 (2008)). CTLA-4 signals suppress both CD4⁺and CD8⁺ T cell responses via a tyrosine-based inhibitory motif (Egen JG, et al. Immunity. 16:23-35 (2002); Egen J G, et al. Nat Immunol.3:611-8 (2002)).

CTLA-4 blockade has antitumor activity in mice, and important effects onbreaking tolerance (Egen J G, et al. Nat Immunol. 3:611-8 (2002); LeachD R, et al. Science. 271:1734-6 (1996); Quezada S A, et al. J ClinInvest. 116:1935-45 (2006); Chen L, et al. Cell. 71:1093-102 (1992); vanElsas A, et al. J Exp Med. 190:355-66 (1999)). In experiments with B16melanoma, a therapeutic effect induced by CTLA-4 blockade with a vaccinewas associated with development of autoimmune vitiligo, suggesting thatexpansion of T cells recognizing melanocyte lineage antigens wasassociated with the therapeutic effect. Several autoimmune diseases wereassociated with single nucleotide polymorphisms in the CTLA-4 gene,including hypothyroidism and type I diabetes (Ueda H, et al. Nature.423:506-11 (2003)). In CTLA-4^(−/−) knock-out mice, expansion oflymphocytes with diffuse lymphadenopathy and lymphoid infiltration ofdifferent organs occurs, consistent with a generalized expansion of Tcells (Waterhouse P, et al. Science. 270:985-8 (1995)). Similarly, bothpreclinical and clinical data indicate that CTLA-4 blockade results inactivation and expansion of total CD4^(|) and CD8^(|) effector T cells(Wolchok J D, et al. Oncologist. 13 Suppl 4:2-9 (2008)), and breaking ofself-tolerance has been shown in patients, as evidenced by theoccurrence of immune-related adverse events (irAEs) observed with twodifferent CTLA-4 antibodies, ipilimumab (Bristol Myers Squibb,Princeton, N.J., USA) (Maker A V, et al. J Immunol. 175:7746-54 (2005))and tremelimumab (Pfizer, New York, N.Y., USA) (Ribas A, et al.Oncologist. 13 Suppl 4:10-5 (2008)).

Ipilimumab, a fully human, CTLA-4 blocking lgG₁ monoclonal antibodyinduces long-lasting clinical responses in a minority of patients withmetastatic melanoma (Wolchok J D, et al. Lancet Oncol. 11:155-64 (2008);Weber J, et al. Clin Cancer Res. 15:5591-8 (2009); Weber J. CancerImmunol Immunother. 58:823-30 (2009); Fong L, et al. J Clin Oncol.26:5275-83 (2008); Phan G Q, et al. Proc Natl Acad Sci US A. 100:8372-7(2003); O'Day S J, et al. Ann Oncol. 21:1712-7 (2010)). Ipilimumab, withor without a gp100 peptide vaccine, compared with gp100 vaccine alone,improved overall survival (OS) in patients with previously treatedmetastatic melanoma (Hodi F S, et al. NEngl J Med. 363:711-23 (2010)).Ipilimumab combined with dacarbazine improved overall survival inpreviously untreated patients compared to dacarbazine alone (Robert C,et al. NEngl J Med. 364:2517-26 (2011)). These were the first randomizedPhase III trials to demonstrate a significant survival impact forpatients with metastatic melanoma, yet few studies have documentedpharmacodynamic markers of the impact of ipilimumab. An increase in theabsolute lymphocyte count (ALC) after 2 or 3 doses of the drug at weeks4 and 7 has been documented (Yuan J, et al. Cancer Immun. 10:1 (2010)),and may correlate with an improved outcome; increased CD4⁺ HLA-DR⁺ Tcells have been shown in several studies to occur after ipilimumabtherapy (Sanderson K, et al. J Clin Oncol. 23:741-50 (2005); Sarnaik AA, et al. Clin Cancer Res. 17(4):896-906 (2010)); in several smallcohort studies of brief duration, ipilimumab treatment increased thefrequency of CD4⁺ ICOS^(hi) T cells in tumors and in the circulation,and also induced antibody reactivity against the cancer-testis antigenNY-ESO-1 Carthon B C, et al. Clin Cancer Res. 16:2861-71 (2010); Chen H,et al. Proc Natl Acad Sci US A. 106:2729-34 (2009)). CTLA-4 abrogatingantibodies do not alter vaccine specific immune responses (Attia P, etal. J Clin Oncol. 23:6043-53 (2005)) and even when administered with apeptide vaccine, tumor antigen specific responses were only modestlyincreased (Attia P, et al. J Clin Oncol. 23:6043-53 (2005); Ribas A, etal. Clin Cancer Res. 15:390-9 (2009)). Recall responses to viral andother antigens were not altered by ipilimumab. In patients receivinganother CTLA-4 abrogating antibody, tremelimumab, the ratio ofintratumoral T cells to FoxP3 positive T regulatory cells was increasedafter treatment only in regressing lesions (Ribas A, et al. Clin CancerRes. 15:390-9 (2009)), suggesting a therapeutic impact of CTLA-4abrogation on T cells infiltrating the tumor. The same investigatorsalso demonstrated that peripheral blood Th17 cells were induced bytremelimumab (von Euw E, et al. J Transl Med. 7:35 (2009)), and thatcertain signaling pathways downstream of the TCR and cytokine receptorwere also influenced by CTLA-4 blockade, such as increased pp38, pSTAT1and pSTAT3, and decreased pLck, pERK1/2 and pSTAT5 levels (Comin-AnduixB, et al. PLoS One. 5:e12711 (2010)). CTLA-4 blockade also induced cellproliferation in the spleen, a secondary lymphoid organ, shown bymolecular imaging with the PET probe 18F-FLT (Ribas A, et al. J NuclMed. 51:340-6 (2010)). They also reported significantly increasedintratumoral CD8^(|) T cell infiltration and CD4^(|) T cellsinfiltration, demonstrated the activation of lymphocytes within tumorsites, as increase of HLA-DR and CD45RO double positive cells in posttremelimumab biopsies (Huang R R, et al. Clin Cancer Res. 17:4101-9(2011)) and increased expression of FoxP3.

To date, the precise molecular basis and mechanisms of action ofipilimumab have not been documented systematically in vivo. There is acritical need for biomarkers of the effects of ipilimumab as well aspredictive biomarkers for clinical outcome and induction of irAE.

PD-1 blocking antibody (BMS-936558) therapy has shown antitumor activityand clinical benefit in melanoma patients. The precise molecular basisand mechanisms of PD-1 blockade in vivo have not been documented andthere are few biomarker associated with clinical benefit. There is acritical need for biomarkers of the effects of PD-1 blockade treatmentas well as predictive biomarkers for clinical outcome.

SUMMARY OF THE INVENTION

Biomarkers have been identified for predicting the efficacy, risk ofrelapse, risk of an immune related adverse event (irAE), or combinationthereof for a CTLA-4 blockade treatment, such as ipilimumab, in asubject with a cancer, such as melanoma. Methods are therefore disclosedthat involve assaying peripheral blood mononuclear cells (PBMCs) from asubject diagnosed with a cancer, such as a melanoma, for expression ofone or more of the following biomarkcrs: CD8, Ki67, comcsodcrmin(EOMES), transforming growth factor beta receptor III (TGFβR3), C—Cchemokine receptor type 7 (CCR7), CD4, CD71, and CD109. For example,expression can be assayed for one or more of the following biomarkers:CD8, Ki67, eomesodermin (EOMES), transforming growth factor betareceptor III (TGFβR3), and C—C chemokine receptor type 7 (CCR7). Forexample, the method can involve assaying the PBMCs for the presence of(a) CD8, Ki67, and EOMES; (b) CD8 and EOMES; (c) CD8 and TGFβR3; (d) CD4and CCR7; (e) CD4, Ki67, and EOMES; (f) CD4 and CD71; or (g) CD8 andCD109. For example, the method can involve assaying the PBMCs for thepresence of (a) CD8, Ki67, and EOMES; (b) CD8 and EOMES; (c) CD8 andTGFβR3; (d) CD4 and CCR7; or (e) CD4, Ki67, and EOMES.

Also disclosed are methods of treating a subject diagnosed with melanomawith a CTLA-4 blockade treatment if the expression level of one or moreof the biomarkers listed in FIGS. 11, 12, 13, and 14 are at or beyond athreshold level, such as a level that is greater than or less than(depending on the marker) a reference level. For example, the ratio ofresponder (NED or non-irAE) to nonresponder (Relapse or irAE) can beused as, or to define a threshold level or a reference level.

Also disclosed are methods of not treating a subject with a CTLA-4blockade treatment if the expression level of one or more of thebiomarkers listed in Tables 10, 11, 12, and 13 are at or beyond athreshold level, such as a level that is greater than or less than(depending on the marker) a reference level. For example, the ratio ofresponder (NED or non-irAE) to nonresponder (Relapse or irAE) can beused as, or to define a threshold level or a reference level.

In some embodiments, the frequency of Ki67⁺EOMES⁺CD8⁺ T cells in CD8⁺ Tcells in the PBMCs is inversely associated with the risk of relapseafter CTLA-4 blockade treatment. For example, a frequency ofKi67⁺EOMES⁺CD8⁺ T cells in CD8⁺ T cells in the PBMCs less than about2.11%, preferably less than about 2.0%, more preferably less than about1.8%, most preferably less than about 1.6%, is an indication that thesubject is at risk of relapse after CTLA-4 blockade treatment. Likewise,a frequency of Ki67⁺EOMES⁺CD8⁺ T cells in CD8⁺ T cells in the PBMCsgreater than about 2.2%, preferably greater than about 2.3%, morepreferably greater than about 2.4%, most preferably greater than about2.5%, is an indication that the subject will have relapse free survivalafter CTLA-4 blockade treatment.

In some embodiments, the frequency of EOMES⁺CD8⁺ T cells in CD8⁺ T cellsin the PBMCs is inversely associated with the risk of relapse afterCTLA-4 blockade treatment. For example, a frequency of EOMES⁺CD8⁺ Tcells in CD8⁺ T cells in the PBMCs less than about 55.6%, preferablyless than about 50%, more preferably less than about 45%, mostpreferably less than about 40%, is an indication that the subject is atrisk of relapse after CTLA-4 blockade treatment. Likewise, a frequencyof EOMES⁺CD8⁺ T cells in CD8⁺ T cells in the PBMCs greater than about56%, preferably greater than about 57%, more preferably greater thanabout 59%, most preferably greater than about 61% is an indication thatthe subject will have relapse free survival after CTLA-4 blockadetreatment.

In some embodiments, the expression level of CCR7 on CD4⁺ T cells in thePBMCs is directly associated with the risk of relapse after CTLA-4blockade treatment. For example, an expression level of CCR7 on CD4⁺ Tcells in the PBMCs greater than about 2402, preferably greater thanabout 2500, more preferably greater than about 2700, most preferablygreater than about 2900, is an indication that the subject is at risk ofrelapse after CTLA-4 blockade treatment. Likewise, an expression levelof CCR7 on CD4⁺ T cells in the PBMCs less than about 2400, preferablyless than about 2300, more preferably less than about 2100, mostpreferably less than about 1900, is an indication that the subject willhave relapse free survival after CTLA-4 blockade treatment.

In some embodiments, the frequency of Ki67⁺EOMES⁺CD4⁺ T cells in CD4⁺ Tcells in the PBMCs is inversely associated with the risk of an immunerelated adverse event (irAE) after CTLA-4 blockade treatment. Forexample, a frequency of Ki67⁺EOMES⁺CD4⁺ T cells in CD4⁺ T cells in thePBMCs less than about 0.446%, preferably less than about 0.4%, morepreferably less than about 0.35%, most preferably less than about 0.3%,is an indication that the subject is at risk of an immune relatedadverse event (irAE) after CTLA-4 blockade treatment. Likewise, afrequency of Ki67⁺EOMES⁺CD4⁺ T cells in CD4⁺ T cells in the PBMCsgreater than about 0.45%, preferably greater than about 0.5%, morepreferably greater than about 0.6%, most preferably greater than about0.7%, is an indication that the subject will not have an irAE afterCTLA-4 blockade treatment.

In some embodiments, the expression level of TGFβR3 on CD8⁺ T cells inthe PBMCs is directly associated with risk of an immune related adverseevent (irAE) after CTLA-4 blockade treatment. For example, an expressionlevel of TGFβR3 on CD8⁺ T cells in the PBMCs greater than about 527,preferably greater than about 600, more preferably greater than about700, most preferably greater than about 1000, is an indication that thesubject is at risk of an immune related adverse event (irAE) afterCTLA-4 blockade treatment. Likewise, an expression level of TGFβR3 onCD8⁺ T cells in the PBMCs less than about 525, preferably less thanabout 500, more preferably less than about 450, most preferably lessthan about 400, is an indication that the subject will not have animmune related adverse event (irAE) after CTLA-4 blockade treatment.

In some embodiments, the expression level of CD71 on CD4⁺ T cells in thePBMCs is inversely associated with risk of an immune related adverseevent (irAE) after CTLA-4 blockade treatment.

In some embodiments, the frequency of CD109⁺CD8⁺ T cells in CD8⁺ T cellsin the PBMCs is directly associated with the risk of relapse afterCTLA-4 blockade treatment. For example, a frequency of CD109⁺CD8⁺ Tcells in CD8⁺ T cells in the PBMCs greater than about 0.7315%,preferably greater than about 0.8%, more preferably greater than about1.0%, most preferably greater than about 2.0%, is an indication that thesubject is at risk of relapse after CTLA-4 blockade treatment. Likewise,a frequency of CD109⁺CD8⁺ T cells in CD8⁺ T cells in the PBMCs less thanabout 0.7316%, preferably less than about 0.7%, more preferably lessthan about 0.6%, most preferably less than about 0.5% is an indicationthat the subject will have relapse free survival after CTLA-4 blockadetreatment.

In some embodiments, the frequency of CD71⁺CD4⁺ T cells in CD4⁺ T cellsin the PBMCs is inversely associated with the risk of an immune relatedadverse event (irAE) after CTLA-4 blockade treatment. For example, afrequency of CD71⁺CD4⁺ T cells in CD4⁺ T cells in the PBMCs less thanabout 2.80%, preferably less than about 2.5%, more preferably less thanabout 2.0%, most preferably less than about 1.5%, is an indication thatthe subject is at risk of an immune related adverse event (irAE) afterCTLA-4 blockade treatment. Likewise, a frequency of CD71⁺CD4⁺ T cells inCD4⁺ T cells in the PBMCs greater than about 2.8%, preferably greaterthan about 3.0%, more preferably greater than about 3.5%, mostpreferably greater than about 4%, is an indication that the subject willnot have an irAE after CTLA-4 blockade treatment.

The disclosed method can further involve using the biomarkers to selectsubjects suitable for treatment with a CTLA-4 blockade agent, such asipilimumab or tremelimumab, or a treatment other than a CTLA-4 blockadetreatment, such as a neoplastic agent, or a combination of CTLA-4blockade agent and another treatment.

For example, in some embodiments, the method involves selecting asubject for CTLA-4 blockade treatment, another treatment, or acombination thereof if (a) if the frequency of Ki67⁺EOMES⁺CD8⁺ T cellsin the subject's CD8⁺ T cells is at least about 2.2%, preferably atleast about 2.3%, more preferably at least about 2.4%, most preferablyat least about 2.5%; (b) if the frequency of EOMES⁺CD8⁺ T cells in thesubject's CD8⁺ T cells is at least 56%, preferably at least about 57%,more preferably at least about 59%, most preferably at least about 61%;(c) if the expression level of CCR7 on CD4⁺ T cells in the subject islower than about 2400, preferably lower than about 2300, more preferablylower than about 2100, most preferably lower than about 1900; (d) if thefrequency of Ki67⁺EOMES⁺CD4⁺ T cells in the subject's CD4⁺ T cells is atleast about 0.45%, preferably at least about 0.5%, more preferably atleast about 0.6%, most preferably at least about 0.7%; (e) if theexpression level of TGFβR3 on CD8⁺ T cells in the subject is lower thanabout 525; preferably lower than about 500, more preferably lower thanabout 450, most preferably lower than about 400; (f) the expressionlevel of CD109 on CD8⁺ T cells is less than 0.7316%, preferably lessthan about 0.7%, more preferably less than about 0.6%, most preferablyless than about 0.5%; (g) the frequency of CD71⁺CD4⁺ T cells in CD4⁺ Tcells in the PBMCs is at least 2.80%, preferably greater than about3.0%, more preferably greater than about 3.5%, most preferably greaterthan about 4%; or any combination thereof.

Therefore, in these embodiments, the subject is not treated with CTLA-4blockade treatment, is treated with another treatment, or a combinationthereof if (a) the frequency of Ki67⁺EOMES⁺CD8⁺ T cells is less thanabout 2.11%, preferably less than about 2.0%, more preferably less thanabout 1.8%, most preferably less than about 1.6%; (b) the frequency ofEOMES⁺CD8⁺ T cells is less than about 55.6%, preferably less than about50%, more preferably less than about 45%, most preferably less thanabout 40%; (c) the expression level of CCR7 on CD4⁺ T cells is greaterthan about 2402, preferably greater than about 2500, more preferablygreater than about 2700, most preferably greater than about 2900; (d)the frequency of Ki67⁺EOMES⁺CD4⁺ T cells is less than about 0.446%,preferably less than about 0.4%, more preferably less than about 0.35%,most preferably less than about 0.3%; (e) the expression level of TGFβR3on CD8⁺ T cells is greater than about 527, preferably greater than about600, more preferably greater than about 700, most preferably greaterthan about 1000; (f) the frequency of CD109⁺CD8⁺ T cells in CD8⁺ T cellsis greater than about 0.7315%, preferably greater than about 0.8%, morepreferably greater than about 1.0%, most preferably greater than about2.0%; (g) the frequency of CD71⁺CD4⁺ T cells in CD4⁺ T cells is lessthan about 2.80%/a, preferably less than about 2.5%, more preferablyless than about 2.0%, most preferably less than about 1.5% or anycombination thereof.

Methods of treating subjects diagnosed with melanoma are also disclosedthat involve treating the subject with a CTLA-4 blockade treatment,another treatment, or a combination thereof (a) if the frequency ofKi67⁺EOMES⁺CD8⁺ T cells in the subject's CD8⁺ T cells is at least about2.2%, preferably at least about 2.3%, more preferably at least about2.4%, most preferably at least about 2.5%; (b) if the frequency ofEOMES⁺CD8⁺ T cells in the subject's CD8⁺ T cells is at least 56%,preferably at least about 57%, more preferably at least about 59%; (c)if the expression level of CCR7 on CD4⁺ T cells in the subject is lowerthan about 2400, preferably lower than about 2300, more preferably lowerthan about 2100, most preferably lower than about 1900; (d) if thefrequency of Ki67⁺EOMES⁺CD4⁺ T cells in the subject's CD4⁺ T cells is atleast about 0.45%, preferably at least about 0.5%, more preferably atlcast about 0.6%, most preferably at least about 0.7%; (e) if theexpression level of TGFβR3 on CD8⁺ T cells in the subject is lower thanabout 525; preferably lower than about 500, more preferably lower thanabout 450, most preferably lower than about 400; (f) the expressionlevel of CD109 on CD8⁺ T cells is less than 0.7316%, preferably lessthan about 0.7%, more preferably less than about 0.6%, most preferablyless than about 0.5%; (g) the frequency of CD71⁺CD4⁺ T cells in CD4⁺ Tcells in the PBMCs is at least 2.80%, preferably greater than about3.0%, more preferably greater than about 3.5%, most preferably greaterthan about 4%; or any combination thereof.

Biomarkers have been identified for predicting the efficacy and clinicalbenefit for a PD-1 blockade treatment, such as a PD-1 blocking antibody,in a subject with a cancer, such as melanoma. Methods are thereforedisclosed that involve assaying peripheral blood mononuclear cells(PBMCs) from a subject diagnosed with a cancer, such as a melanoma, forexpression of one or more of the biomarkers listed in Tables 8 and 9.

Disclosed are methods comprising assaying peripheral blood mononuclearcells (PBMCs) from a subject diagnosed with melanoma for expression ofCD8 and one or a combination of markers in Tables 8 and 9, where,depending on the marker, the level of expression of certain markers onCD8⁺ T cells in the PBMCs of the subject prior to PD-1 blockadetreatment or a change in the expression level of certain markers on CD8⁺T cells in the PBMCs following PD-1 blockade treatment indicate theexpected clinical benefit of PD-1 blockade treatment of the subject.

Also disclosed are methods of treating a subject diagnosed with melanomawith a PD-1 blockade treatment if the expression level of one or more ofthe biomarkers listed in Tables 8 and 9 are at or beyond a thresholdlevel, such as a level that is greater than or less than (depending onthe marker) a reference level.

Also disclosed are methods of treating a subject diagnosed with melanomawith a treatment other than a PD-1 blockade treatment, or with both aPD-1 blockade treatment and a different treatment, if the expressionlevel of one or more of the biomarkers listed in Tables 8 and 9 are ator beyond a threshold level, such as a level that is greater than orless than (depending on the marker) a reference level.

In some forms of the methods, the PBMCs can be assayed prior to PD-1blockade treatment of the subject. Expression levels measured prior toPD-1 blockade treatment can be referred to as baseline expression. Insome forms of the methods, the expression prior the PD-1 blockadetreatment is compared to a reference level of expression.

In some forms of the methods, the PBMCs can also be assayed followingPD-1 blockade treatment of the subject. Expression following the PD-1blockade treatment can be compared to the expression prior to the PD-1blockade treatment. Depending on the marker, a change in the expressionlevel of certain markers on CD8⁺ T cells in the PBMCs following PD-1blockade treatment indicate the expected clinical benefit of PD-1blockade treatment of the subject. In some forms, a change in expressionlevel can indicate that PD-1 blockade treatment should be continued withthe subject.

Also disclosed are methods of selecting a subject for PD-1 blockadetreatment if the expression level of one or more of the biomarkerslisted in Tables 8 and 9 are at or beyond a threshold level, such as alevel that is greater than or less than (depending on the marker) areference level.

Also disclosed are methods of not treating a subject with a PD-1blockade treatment if the expression level of one or more of thebiomarkers listed in Tables 8 and 9 are at or beyond a threshold level,such as a level that is greater than or less than (depending on themarker) a reference level.

In some forms of the methods, the PD-1 blockade treatment can betreatment with a PD-1 blocking antibody. In some forms of the methods,the PD-1 blockade treatment can be treatment with PD-1 blocking antibodyBMS-936558 or BMS-936559.

For example, expression of one or a combination of Phosphatase andtensin homolog (PTEN), GATA zinc finger domain containing 1 (GATAD1),Natural killer-tumor recognition sequence (NKTR), CD276 molecule (B7-H3)(CD276), NOL1/NOP2/Sun domain family, member 6 (NSUN6), Cell divisioncycle and apoptosis regulator 1 (CCAR1), SET and MYND domain containing2 (SMYD2), F-box protein 9 (FBXO9), Non-protein coding RNA153(NCRNA00153), Neutral sphingomyelinase activation associated factor(NSMAF), Pyrimidinergic receptor P2Y, G-protein coupled, 4 (P2RY4), RESTcorcprcssor 2 (RCOR2), Negative regulator of ubiquitin-like proteins 1(NUB1), Fc receptor-like 2 (FCRL2), Nuclear casein kinase,cyclin-dependent kinase substrate 1 (NUCKS1), SHC transforming protein 2(SHC2), Stearoyl-CoA desaturase 5 (SCD5), Ubiquitin specific peptidase9, Y-linked (USP9Y), Heat shock 60 kDa protein 1 (chaperonin) (HSPD1),Cytokine inducible SH2-containing protein (CISH), Kruppel-like factor 12(KLF12), Neutrophil cytosolic factor 4, 40 kDa (NCF4), Splicing factor,arginine/serine-rich 7, 35 kDa (SFRS7), CDC14 cell division cycle 14homolog A (S. cerevisiae) (CDC14A), Mitogen-activated protein kinasekinase 5 (MAP2K5), Interleukin 11 receptor, alpha (IL11RA),Myeloid/lymphoid or mixed-lineage leukemia (MLL), HSPB (heat shock 27kDa) associated protein 1 (HSPBAP1), Mitogen-activated protein kinasekinase kinase kinase 4 (MAP4K4), Helicase, lymphoid-specific (HELLS),C1q and tumor necrosis factor related protein 3 (C1QTNF3),Ubiquitin-conjugating enzyme E2D 1 (UBE2D1), Fas apoptotic inhibitorymolecule 3 (FAIM3), Interferon-induced protein 44 (IFI44), H2B histonefamily, member M (H2BFM), GATA zinc finger domain containing 1 (GATAD1),Caspase 8, apoptosis-related cysteine peptidase (CASP8), Suppressor ofcytokine signaling 1 (SOCS1), Programmed cell death 6 (PDCD6), LAG1homolog, ceramide synthase 6 (LASS6), Growth arrestDNA-damage-inducible, beta (GADD45B), Early growth response 2 (EGR2),Early growth response 1 (EGR1), Growth arrest, DNA-damage-inducible, g(GADD45G), CDC28 protein kinase regulatory subunit 2 (CKS2),Cyclin-dependent kinase inhibitor 1C (CDKN1C), Immediate early response5 (IER5), Inhibitor of growth family, member 1 (ING1), Interferoninduced with helicase C domain 1 (IFIH1), Interferon, gamma (IFNG),Immediate early response 5-like (IER5L), Cancer/testis antigen 1(NY-ESO-1), and Melanoma antigen recognized by T-cells 1 (MART-1) can bedetected, measured, assayed, assessed, etc.

The levels of expression of the markers can indicate the efficacy,predicted efficacy, likelihood of clinical benefit, prognosis, etc., ofa subject treated with a PD-1 blockade treatment. For example, theexpression level of PTEN on CD8⁺ T cells in the PBMCs is positivelyassociated with the expected clinical benefit of PD-1 blockade treatmentof the subject; the expression level of GATAD1 on CD8⁺ T cells in thePBMCs is positively associated with the expected clinical benefit ofPD-1 blockade treatment of the subject; the expression level of NKTR onCD8⁺ T cells in the PBMCs is positively associated with the expectedclinical benefit of PD-1 blockade treatment of the subject; theexpression level of CD276 on CD8⁺ T cells in the PBMCs is negativelyassociated with the expected clinical benefit of PD-1 blockade treatmentof the subject; the expression level of NSUN6 on CD8⁺ T cells in thePBMCs is positively associated with the expected clinical benefit ofPD-1 blockade treatment of the subject; the expression level of CCAR1 onCD8⁺ T cells in the PBMCs is positively associated with the expectedclinical benefit of PD-1 blockade treatment of the subject; theexpression level of SMYD2 on CD8⁺ T cells in the PBMCs is positivelyassociated with the expected clinical benefit of PD-1 blockade treatmentof the subject; the expression level of FBXO9 on CD8⁺ T cells in thePBMCs is positively associated with the expected clinical benefit ofPD-1 blockade treatment of the subject; the expression level ofNCRNA00153 on CD8⁺ T cells in the PBMCs is positively associated withthe expected clinical benefit of PD-1 blockade treatment of the subject;the expression level of NSMAF on CD8⁺ T cells in the PBMCs is positivelyassociated with the expected clinical benefit of PD-1 blockade treatmentof the subject; the expression level of P2RY4 on CD8⁺ T cells in thePBMCs is negatively associated with the expected clinical benefit ofPD-1 blockade treatment of the subject; the expression level of RCOR2 onCD8⁺ T cells in the PBMCs is negatively associated with the expectedclinical benefit of PD-1 blockade treatment of the subject; theexpression level of NUB1 on CD8⁺ T cells in the PBMCs is negativelyassociated with the expected clinical benefit of PD-1 blockade treatmentof the subject; the expression level of FCRL2 on CD8⁺ T cells in thePBMCs is negatively associated with the expected clinical benefit ofPD-1 blockade treatment of the subject; the expression level of NUCKS1on CD8⁺ T cells in the PBMCs is negatively associated with the expectedclinical benefit of PD-1 blockade treatment of the subject; theexpression level of SHC2 on CD8⁺ T cells in the PBMCs is negativelyassociated with the expected clinical benefit of PD-1 blockade treatmentof the subject; the expression level of SCD5 on CD8⁺ T cells in thePBMCs is negatively associated with the expected clinical benefit ofPD-1 blockade treatment of the subject; the expression level of USP9Y onCD8⁺ T cells in the PBMCs is positively associated with the expectedclinical benefit of PD-1 blockade treatment of the subject; theexpression level of HSPD1 on CD8⁺ T cells in the PBMCs is positivelyassociated with the expected clinical benefit of PD-1 blockade treatmentof the subject; the expression level of CISH on CD8⁺ T cells in thePBMCs is positively associated with the expected clinical benefit ofPD-1 blockade treatment of the subject; the expression level of KLF12 onCD8⁺ T cells in the PBMCs is positively associated with the expectedclinical benefit of PD-1 blockade treatment of the subject; theexpression level of NCF4 on CD8⁺ T cells in the PBMCs is positivelyassociated with the expected clinical benefit of PD-1 blockade treatmentof the subject; the expression level of SFRS7 on CD8^(|) T cells in thePBMCs is positively associated with the expected clinical benefit ofPD-1 blockade treatment of the subject; the expression level of CDC14Aon CD8⁺ T cells in the PBMCs is positively associated with the expectedclinical benefit of PD-1 blockade treatment of the subject; theexpression level of MAP2K5 on CD8⁺ T cells in the PBMCs is positivelyassociated with the expected clinical benefit of PD-1 blockade treatmentof the subject; the expression level of IL11RA on CD8⁺ T cells in thePBMCs is positively associated with the expected clinical benefit ofPD-1 blockade treatment of the subject; the expression level of MLL onCD8⁺ T cells in the PBMCs is positively associated with the expectedclinical benefit of PD-1 blockade treatment of the subject; theexpression level of HSPBAP1 on CD8^(|) T cells in the PBMCs ispositively associated with the expected clinical benefit of PD-1blockade treatment of the subject; the expression level of MAP4K4 onCD8⁺ T cells in the PBMCs is positively associated with the expectedclinical benefit of PD-1 blockade treatment of the subject; theexpression level of HELLS on CD8⁺ T cells in the PBMCs is positivelyassociated with the expected clinical benefit of PD-1 blockade treatmentof the subject; the expression level of C1QTNF3 on CD8⁺ T cells in thePBMCs is positively associated with the expected clinical benefit ofPD-1 blockade treatment of the subject; the expression level of UBE2D1on CD8⁺ T cells in the PBMCs is positively associated with the expectedclinical benefit of PD-1 blockade treatment of the subject; theexpression level of FAIM3 on CD8⁺ T cells in the PBMCs is positivelyassociated with the expected clinical benefit of PD-1 blockade treatmentof the subject; the expression level of IFI44 on CD8⁺ T cells in thePBMCs is positively associated with the expected clinical benefit ofPD-1 blockade treatment of the subject; the expression level of H2BFM onCD8⁺ T cells in the PBMCs is positively associated with the expectedclinical benefit of PD-1 blockade treatment of the subject; theexpression level of GATAD1 on CD8⁺ T cells in the PBMCs is positivelyassociated with the expected clinical benefit of PD-1 blockade treatmentof the subject; the expression level of CASP8 on CD8⁺ T cells in thePBMCs is positively associated with the expected clinical benefit ofPD-1 blockade treatment of the subject; the expression level of SOCS1 onCD8⁺ T cells in the PBMCs is negatively associated with the expectedclinical benefit of PD-1 blockade treatment of the subject; theexpression level of PDCD6 on CD8⁺ T cells in the PBMCs is negativelyassociated with the expected clinical benefit of PD-1 blockade treatmentof the subject; the expression level of NY-ESO-1 on CD8⁺ T cells in thePBMCs is positively associated with the expected clinical benefit ofPD-1 blockade treatment of the subject; the expression level of MART-1on CD8⁺ T cells in the PBMCs is positively associated with the expectedclinical benefit of PD-1 blockade treatment of the subject; an increasein the expression level of LASS6 on CD8⁺ T cells in the PBMCs followingPD-1 blockade treatment is positively associated with the expectedclinical benefit of PD-1 blockade treatment of the subject; a decreasein the expression level of GADD45B on CD8⁺ T cells in the PBMCsfollowing PD-1 blockade treatment is positively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject; adecrease in the expression level of EGR2 on CD8⁺ T cells in the PBMCsfollowing PD-1 blockade treatment is positively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject; adecrease in the expression level of EGR1 on CD8⁺ T cells in the PBMCsfollowing PD-1 blockade treatment is positively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject; adecrease in the expression level of GADD45G on CD8⁺ T cells in the PBMCsfollowing PD-1 blockade treatment is positively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject; adecrease in the expression level of CKS2 on CD8⁺ T cells in the PBMCsfollowing PD-1 blockade treatment is positively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject; adecrease in the expression level of CDKN1C on CD8⁺ T cells in the PBMCsfollowing PD-1 blockade treatment is positively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject; adecrease in the expression level of IER5 on CD8⁺ T cells in the PBMCsfollowing PD-1 blockade treatment is positively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject; adecrease in the expression level of ING1 on CD8⁺ T cells in the PBMCsfollowing PD-1 blockade treatment is positively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject; adecrease in the expression level of IFIH1 on CD8⁺ T cells in the PBMCsfollowing PD-1 blockade treatment is positively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject; adecrease in the expression level of IFNG on CD8⁺ T cells in the PBMCsfollowing PD-1 blockade treatment is positively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject; adecrease in the expression level of IER5L on CD8⁺ T cells in the PBMCsfollowing PD-1 blockade treatment is positively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject; anincrease in the expression level of NY-ESO-1 on CD8⁺ T cells in thePBMCs following PD-1 blockade treatment is positively associated withthe expected clinical benefit of PD-1 blockade treatment of the subject;an increase in the expression level of MART-1 on CD8^(|) T cells in thePBMCs following PD-1 blockade treatment is positively associated withthe expected clinical benefit of PD-1 blockade treatment of the subject;a decrease in the number or frequency of T regulatory cells (Tregs;characterized as FoxP3⁺CD127^(Low) CD25⁺CD4⁺) in CD4⁺ T cells in PBMCsfollowing PD-1 blockade treatment is positively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject; anincrease in the number or frequency of T regulatory cells (Tregs;characterized as FoxP3⁺CD127^(Low)CD25⁺CD4⁺) in CD4⁺ T cells in PBMCsfollowing PD-1 blockade treatment is negatively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject; adecrease in the number or frequency of CTLA-4⁺CD4⁺ cells in CD4⁺ T cellsin PBMCs following PD-1 blockade treatment is positively associated withthe expected clinical benefit of PD-1 blockade treatment of the subject;an increase in the number or frequency of CTLA-4⁺CD4⁺ cells in CD4⁺ Tcells in PBMCs following PD-1 blockade treatment is negativelyassociated with the expected clinical benefit of PD-1 blockade treatmentof the subject; a decrease in the number or frequency of CTLA-4⁺CD8⁺cells in CD8⁺ T cells in PBMCs following PD-1 blockade treatment ispositively associated with the expected clinical benefit of PD-1blockade treatment of the subject; an increase in the number orfrequency of CTLA-4⁺CD8⁺ cells in CD8⁺ T cells in PBMCs following PD-1blockade treatment is negatively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject.

As another example, disclosed are methods of treating a subjectdiagnosed with melanoma with a PD-1 blockade treatment if the expressionlevel of PTEN on CD8⁺ T cells in the PBMCs prior to PD-1 blockadetreatment is more than 1.2 times, preferably more than 1.3 times, mostpreferably more than 1.4 times the reference level of PTEN; theexpression level of GATAD1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.1 times, preferably more than 1.3 times, mostpreferably more than 1.4 times the reference level of GATAD1; theexpression level of NKTR on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.1 times, preferably more than 1.3 times, mostpreferably more than 1.4 times the reference level of NKTR; theexpression level of CD276 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 0.9 times, preferably less than 0.8 times, mostpreferably less than 0.7 times the reference level of CD276; theexpression level of NSUN6 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.4 times, preferably more than 1.7 times, mostpreferably more than 1.9 times the reference level of NSUN6; theexpression level of CCAR1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.2 times, preferably more than 1.5 times, mostpreferably more than 1.7 times the reference level of CCAR1; theexpression level of SMYD2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.2 times, preferably more than 1.4 times, mostpreferably more than 1.6 times the reference level of SMYD2; theexpression level of FBXO9 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.2 times, preferably more than 1.3 times, mostpreferably more than 1.4 times the reference level of FBXO9; theexpression level of NCRNA00153 on CD8⁺ T cells in the PBMCs prior toPD-1 blockade is more than 1.2 times, preferably more than 1.3 times,most preferably more than 1.4 times the reference level of NCRNA00153;the expression level of NSMAF on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.2 times, preferably more than 1.3 times, mostpreferably more than 1.4 times the reference level of NSMAF; theexpression level of P2RY4 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 0.9 times, preferably less than 0.8 times, mostpreferably less than 0.7 times the reference level of P2RY4; theexpression level of RCOR2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 0.9 times, preferably less than 0.8 times, mostpreferably less than 0.7 times the reference level of RCOR2; theexpression level of NUB1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 0.9 times, preferably less than 0.8 times, mostpreferably less than 0.7 times the reference level of NUB1; theexpression level of FCRL2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 0.9 times, preferably less than 0.7 times, mostpreferably less than 0.5 times the reference level of FCRL2; theexpression level of NUCKS1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 0.8 times, preferably less than 0.7 times, mostpreferably less than 0.6 times the reference level of NUCKS1; theexpression level of SHC2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 0.8 times, preferably less than 0.7 times, mostpreferably less than 0.6 times the reference level of SHC2; theexpression level of SCD5 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 0.8 times, preferably less than 0.6 times, mostpreferably less than 0.4 times the reference level of SCD5; thefrequency of NY-ESO-1⁺CD8⁺ cells in CD8⁺ T cells in the PBMCs prior toPD-1 blockade treatment is less than 0.5%, preferably less than 0.4%,most preferably less than 0.3%; the frequency of MART-1⁺CD8⁺ cells inCD8⁺ T cells in the PBMCs prior to PD-1 blockade treatment is less than0.5%, preferably less than 0.4%, most preferably less than 0.3%; or acombination. The subject can also expect a clinical benefit from PD-1blockade treatment at these levels of expression.

As another example, disclosed are methods of treating a subjectdiagnosed with melanoma with a PD-1 blockade treatment if the expressionlevel of LASS6 on CD8⁺ T cells in the PBMCs following the PD-1 blockadetreatment is more than 1.2 times, preferably more than 1.3 times, mostpreferably more than 1.4 times the expression level prior to the PD-1blockade treatment; the expression level of GADD45B on CD8⁺ T cells inthe PBMCs following the PD-1 blockade treatment is less than 0.8 times,preferably less than 0.7 times, most preferably less than 0.6 times theexpression level prior to the PD-1 blockade treatment; the expressionlevel of EGR2 on CD8⁺ T cells in the PBMCs following the PD-1 blockadetreatment is less than 0.8 times, preferably less than 0.7 times, mostpreferably less than 0.6 times the expression level prior to the PD-1blockade treatment; the expression level of EGR1 on CD8⁺ T cells in thePBMCs following the PD-1 blockade treatment is less than 0.7 times,preferably less than 0.4 times, most preferably less than 0.2 times theexpression level prior to the PD-1 blockade treatment; the expressionlevel of GADD45G on CD8⁺ T cells in the PBMCs following the PD-1blockade treatment is less than 0.9 times, preferably less than 0.8times, most preferably less than 0.7 times the expression level prior tothe PD-1 blockade treatment; the expression level of CKS2 on CD8⁺ Tcells in the PBMCs following the PD-1 blockade treatment is less than0.9 times, preferably less than 0.8 times, most preferably less than 0.7times the expression level prior to the PD-1 blockade treatment; theexpression level of CDKN1C on CD8⁺ T cells in the PBMCs following thePD-1 blockade treatment is less than 0.9 times, preferably less than 0.8times, most preferably less than 0.7 times the expression level prior tothe PD-1 blockade treatment; the expression level of IER5 on CD8⁺ Tcells in the PBMCs following the PD-1 blockade treatment is less than0.9 times, preferably less than 0.8 times, most preferably less than 0.7times the expression level prior to the PD-1 blockade treatment; thefrequency of NY-ESO-1⁺CD8⁺ cells in CD8⁺ T cells in the PBMCs followingthe PD-1 blockade treatment is less than 0.7 times, preferably less than0.6 times, most preferably less than 0.5 times the frequency prior tothe PD-1 blockade treatment; the frequency of MART-1⁺CD8⁺ cells in CD8⁺T cells in the PBMCs following the PD-1 blockade treatment is less than0.7 times, preferably less than 0.6 times, most preferably less than 0.5times the frequency prior to the PD-1 blockade treatment; the number orfrequency of T regulatory cells (Tregs; characterized asFoxP3⁺CD127^(Low)CD25⁺CD4⁺) in CD4⁺ T cells in PBMCs following the PD-1blockade treatment is less than 1.0 times, preferably less than 0.9times, most preferably less than 0.8 times the number or frequency ofTregs in CD4⁺ T cells in PBMCs prior to the PD-1 blockade treatment; thenumber or frequency of CTLA-4⁺CD4⁺ cells in CD4⁺ T cells in PBMCsfollowing the PD-1 blockade treatment is less than 1.0 times, preferablyless than 0.9 times, most preferably less than 0.8 times the number orfrequency of CTLA-4⁺CD4⁺ cells in CD4⁺ T cells in PBMCs prior to thePD-1 blockade treatment; the number or frequency of CTLA-4⁺CD8⁺ cells inCD8⁺ T cells in PBMCs following the PD-1 blockade treatment is less than1.0 times, preferably less than 0.9 times, most preferably less than 0.8times the number or frequency of CTLA-4⁺CD8⁺ cells in CD8⁺ T cells inPBMCs prior to the PD-1 blockade treatment; or a combination.

As another example, disclosed are methods of treating a subjectdiagnosed with melanoma with a treatment other than a PD-1 blockadetreatment, or with both a PD-1 blockade treatment and a differenttreatment, if the expression level of PTEN on CD8⁺ T cells in the PBMCsprior to PD-1 blockade treatment is less than 1.1 times, preferably lessthan 1.0 times, most preferably less than 0.9 times the reference levelof PTEN; the expression level of GATAD1 on CD8⁺ T cells in the PBMCsprior to PD-1 blockade is less than 1.0 times, preferably less than 0.9times, most preferably less than 0.8 times the reference level ofGATAD1; the expression level of NKTR on CD8⁺ T cells in the PBMCs priorto PD-1 blockade is less than 1.0 times, preferably less than 0.9 times,most preferably less than 0.8 times the reference level of NKTR; theexpression level of CD276 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.0 times, preferably more than 1.1 times, mostpreferably more than 1.2 times the reference level of CD276; theexpression level of NSUN6 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 1.2 times, preferably less than 1.0 times, mostpreferably less than 0.8 times the reference level of NSUN6; theexpression level of CCAR1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 1.1 times, preferably less than 1.0 times, mostpreferably less than 0.9 times the reference level of CCAR1; theexpression level of SMYD2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 1.1 times, preferably less than 1.0 times, mostpreferably less than 0.9 times the reference level of SMYD2; theexpression level of FBXO9 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 1.1 times, preferably less than 1.0 times, mostpreferably less than 0.9 times the reference level of FBXO9; theexpression level of NCRNA00153 on CD8⁺ T cells in the PBMCs prior toPD-1 blockade is less than 1.1 times, preferably less than 1.0 times,most preferably less than 0.9 times the reference level of NCRNA00153;the expression level of NSMAF on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 1.1 times, preferably less than 1.0 times, mostpreferably less than 0.9 times the reference level of NSMAF; theexpression level of P2RY4 on CD8^(|) T cells in the PBMCs prior to PD-1blockade is more than 1.0 times, preferably more than 1.1 times, mostpreferably more than 1.2 times the reference level of P2RY4; theexpression level of RCOR2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.0 times, preferably more than 1.1 times, mostpreferably more than 1.2 times the reference level of RCOR2; theexpression level of NUB1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.0 times, preferably more than 1.1 times, mostpreferably more than 1.2 times the reference level of NUB1; theexpression level of FCRL2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.0 times, preferably more than 1.1 times, mostpreferably more than 1.2 times the reference level of FCRL2; theexpression level of NUCKS1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 0.9 times, preferably more than 1.0 times, mostpreferably more than 1.1 times the reference level of NUCKS1; theexpression level of SHC2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 0.9 times, preferably more than 1.0 times, mostpreferably more than 1.1 times the reference level of SHC2; theexpression level of SCD5 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 0.9 times, preferably more than 1.0 times, mostpreferably more than 1.1 times the reference level of SCD5; thefrequency of NY-ESO-1⁺CD8⁺ cells in CD8⁺ T cells in the PBMCs prior toPD-1 blockade treatment is more than 0.5%, preferably more than 0.7%,most preferably more than 0.8%; the frequency of MART-1⁺CD8⁺ cells inCD8⁺ T cells in the PBMCs prior to PD-1 blockade treatment is more than0.5%, preferably more than 0.7/o, most preferably more than 0.8%; thenumber or frequency of Tregs in CD4⁺ T cells in PBMCs following PD-1blockade treatment is more than 1.0 times, preferably more than 1.1times, most preferably more than 1.2 times the number or frequency ofTregs in CD4⁺ T cells in PBMCs prior to the PD-1 blockade treatment; thenumber or frequency of CTLA-4⁺CD4⁺ cells in CD4⁺ T cells in PBMCsfollowing the PD-1 blockade treatment is more than 1.0 times, preferablymore than 1.1 times, most preferably more than 1.2 times the number orfrequency of CTLA-4⁺CD4⁺ cells in CD4⁺ T cells in PBMCs prior to thePD-1 blockade treatment; the number or frequency of CTLA-4⁺CD8⁺ cells inCD8⁺ T cells in PBMCs following the PD-1 blockade treatment is more than1.0 times, preferably more than 1.1 times, most preferably more than 1.2times the number or frequency of CTLA-4⁺CD8⁺ cells in CD8⁺ T cells inPBMCs prior to the PD-1 blockade treatment; or a combination. Thesubject can also expect a clinical benefit from PD-1 blockade treatmentat these levels of expression.

As another example, disclosed are methods of treating a subjectdiagnosed with melanoma with a treatment other than a PD-1 blockadetreatment, or with both a PD-1 blockade treatment and a differenttreatment, if the expression level of LASS6 on CD8⁺ T cells in the PBMCsfollowing the PD-1 blockade treatment is less than 1.1 times, preferablyless than 1.0 times, most preferably less than 0.9 times the expressionlevel prior to the PD-1 blockade treatment; the expression level ofGADD45B on CD8^(|) T cells in the PBMCs following the PD-1 blockadetreatment is more than 0.9 times, preferably more than 1.0 times, mostpreferably more than 1.1 times the expression level prior to the PD-1blockade treatment; the expression level of EGR2 on CD8⁺ T cells in thePBMCs following the PD-1 blockade treatment is more than 0.9 times,preferably more than 1.0 times, most preferably more than 1.1 times theexpression level prior to the PD-1 blockade treatment; the expressionlevel of EGR1 on CD8⁺ T cells in the PBMCs following the PD-1 blockadetreatment is more than 0.8 times, preferably more than 0.9 times, mostpreferably more than 1.0 times the expression level prior to the PD-1blockade treatment; the expression level of GADD45G on CD8⁺ T cells inthe PBMCs following the PD-1 blockade treatment is more than 1.0 times,preferably more than 1.1 times, most preferably more than 1.2 times theexpression level prior to the PD-1 blockade treatment; the expressionlevel of CKS2 on CD8⁺ T cells in the PBMCs following the PD-1 blockadetreatment is more than 1.0 times, preferably more than 1.1 times, mostpreferably more than 1.2 times the expression level prior to the PD-1blockade treatment; the expression level of CDKN1C on CD8⁺ T cells inthe PBMCs following the PD-1 blockade treatment is more than 1.0 times,preferably more than 1.1 times, most preferably more than 1.2 times theexpression level prior to the PD-1 blockade treatment; the expressionlevel of IER5 on CD8⁺ T cells in the PBMCs following the PD-1 blockadetreatment is more than 1.0 times, preferably more than 1.1 times, mostpreferably more than 1.2 times the expression level prior to the PD-1blockade treatment; the number or frequency of Tregs in CD4⁺ T cells inPBMCs following PD-1 blockade treatment is more than 1.0 times,preferably more than 1.1 times, most preferably more than 1.2 times thenumber or frequency of Tregs in CD4⁺ T cells in PBMCs prior to the PD-1blockade treatment; the number or frequency of CTLA-4⁺CD4⁺ cells in CD4⁺T cells in PBMCs following the PD-1 blockade treatment is more than 1.0times, preferably more than 1.1 times, most preferably more than 1.2times the number or frequency of CTLA-4⁺CD4⁺ cells in CD4⁺ T cells inPBMCs prior to the PD-1 blockade treatment; the number or frequency ofCTLA-4⁺CD8⁺ cells in CD8⁺ T cells in PBMCs following the PD-1 blockadetreatment is more than 1.0 times, preferably more than 1.1 times, mostpreferably more than 1.2 times the number or frequency of CTLA-4⁺CD8⁺cells in CD8⁺ T cells in PBMCs prior to the PD-1 blockade treatment; ora combination. The subject would not expect a clinical benefit from PD-1blockade treatment at these levels of expression.

In some forms of the method, the expression level of PTEN on CD8⁺ Tcells in the PBMCs prior to PD-1 blockade treatment is positivelyassociated with the expected clinical benefit of PD-1 blockade treatmentof the subject; the expression level of GATAD1 on CD8⁺ T cells in thePBMCs prior to PD-1 blockade treatment is positively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject; theexpression level of NKTR on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject; the expression levelof CD276 on CD8⁺ T cells in the PBMCs prior to PD-1 blockade treatmentis negatively associated with the expected clinical benefit of PD-1blockade treatment of the subject; the expression level of NSUN6 on CD8⁺T cells in the PBMCs prior to PD-1 blockade treatment is positivelyassociated with the expected clinical benefit of PD-1 blockade treatmentof the subject; the expression level of CCAR1 on CD8⁺ T cells in thePBMCs prior to PD-1 blockade treatment is positively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject; theexpression level of SMYD2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject; the expression levelof FBXO9 on CD8⁺ T cells in the PBMCs prior to PD-1 blockade treatmentis positively associated with the expected clinical benefit of PD-1blockade treatment of the subject; the expression level of NCRNA00153 onCD8⁺ T cells in the PBMCs prior to PD-1 blockade treatment is positivelyassociated with the expected clinical benefit of PD-1 blockade treatmentof the subject; the expression level of NSMAF on CD8⁺ T cells in thePBMCs prior to PD-1 blockade treatment is positively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject; theexpression level of P2RY4 on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is negatively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject; the expression levelof RCOR2 on CD8⁺ T cells in the PBMCs prior to PD-1 blockade treatmentis negatively associated with the expected clinical benefit of PD-1blockade treatment of the subject; the expression level of NUB1 on CD8⁺T cells in the PBMCs prior to PD-1 blockade treatment is negativelyassociated with the expected clinical benefit of PD-1 blockade treatmentof the subject; the expression level of FCRL2 on CD8⁺ T cells in thePBMCs prior to PD-1 blockade treatment is negatively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject; theexpression level of NUCKS1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is negatively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject; the expression levelof SHC2 on CD8⁺ T cells in the PBMCs prior to PD-1 blockade treatment isnegatively associated with the expected clinical benefit of PD-1blockade treatment of the subject; the expression level of SCD5 on CD8⁺T cells in the PBMCs prior to PD-1 blockade treatment is negativelyassociated with the expected clinical benefit of PD-1 blockade treatmentof the subject; the expression level of USP9Y on CD8⁺ T cells in thePBMCs prior to PD-1 blockade treatment is positively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject; theexpression level of HSPD1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject; the expression levelof CISH on CD8⁺ T cells in the PBMCs prior to PD-1 blockade treatment ispositively associated with the expected clinical benefit of PD-1blockade treatment of the subject; the expression level of KLF12 on CD8⁺T cells in the PBMCs prior to PD-1 blockade treatment is positivelyassociated with the expected clinical benefit of PD-1 blockade treatmentof the subject; the expression level of NCF4 on CD8⁺ T cells in thePBMCs prior to PD-1 blockade treatment is positively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject; theexpression level of SFRS7 on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject; the expression levelof CDC14A on CD8⁺ T cells in the PBMCs prior to PD-1 blockade treatmentis positively associated with the expected clinical benefit of PD-1blockade treatment of the subject; the expression level of MAP2K5 onCD8⁺ T cells in the PBMCs prior to PD-1 blockade treatment is positivelyassociated with the expected clinical benefit of PD-1 blockade treatmentof the subject; the expression level of ILl RA on CD8⁺ T cells in thePBMCs prior to PD-1 blockade treatment is positively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject; theexpression level of MLL on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject; the expression levelof HSPBAP1 on CD8⁺ T cells in the PBMCs prior to PD-1 blockade treatmentis positively associated with the expected clinical benefit of PD-1blockade treatment of the subject; the expression level of MAP4K4 onCD8⁺ T cells in the PBMCs prior to PD-1 blockade treatment is positivelyassociated with the expected clinical benefit of PD-1 blockade treatmentof the subject; the expression level of HELLS on CD8⁺ T cells in thePBMCs prior to PD-1 blockade treatment is positively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject; theexpression level of C1QTNF3 on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject; the expression levelof UBE2D1 on CD8⁺ T cells in the PBMCs prior to PD-1 blockade treatmentis positively associated with the expected clinical benefit of PD-1blockade treatment of the subject; the expression level of FAIM3 on CD8⁺T cells in the PBMCs prior to PD-1 blockade treatment is positivelyassociated with the expected clinical benefit of PD-1 blockade treatmentof the subject; the expression level of IFI44 on CD8⁺ T cells in thePBMCs prior to PD-1 blockade treatment is positively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject; theexpression level of H2BFM on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject; the expression levelof GATAD1 on CD8⁺ T cells in the PBMCs prior to PD-1 blockade treatmentis positively associated with the expected clinical benefit of PD-1blockade treatment of the subject; the expression level of CASP8 on CD8⁺T cells in the PBMCs prior to PD-1 blockade treatment is positivelyassociated with the expected clinical benefit of PD-1 blockade treatmentof the subject; the expression level of SOCS1 on CD8⁺ T cells in thePBMCs prior to PD-1 blockade treatment is negatively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject; theexpression level of PDCD6 on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is negatively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject; the expression levelof NY-ESO-1 on CD8⁺ T cells in the PBMCs prior to PD-1 blockadetreatment is negatively associated with the expected clinical benefit ofPD-1 blockade treatment of the subject; and the expression level ofMART-1 on CD8⁺ T cells in the PBMCs prior to PD-1 blockade treatment isnegatively associated with the expected clinical benefit of PD-1blockade treatment of the subject.

In some forms of the method, the expression prior the PD-1 blockadetreatment is compared to a reference level of expression and anexpression level of PTEN on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment greater than the reference level of PTEN indicatesthat the subject can expect a clinical benefit from PD-1 blockadetreatment; an expression level of GATAD1 on CD8⁺ T cells in the PBMCsprior to PD-1 blockade greater than the reference level of GATAD1indicates that the subject can expect a clinical benefit from PD-1blockade treatment; an expression level of NKTR on CD8⁺ T cells in thePBMCs prior to PD-1 blockade greater than the reference level of NKTRindicates that the subject can expect a clinical benefit from PD-1blockade treatment; an expression level of CD276 on CD8^(|) T cells inthe PBMCs prior to PD-1 blockade lower than the reference level of CD276indicates that the subject can expect a clinical benefit from PD-1blockade treatment; an expression level of NSUN6 on CD8⁺ T cells in thePBMCs prior to PD-1 blockade greater than the reference level of NSUN6indicates that the subject can expect a clinical benefit from PD-1blockade treatment; an expression level of CCAR1 on CD8⁺ T cells in thePBMCs prior to PD-1 blockade greater than the reference level of CCAR1indicates that the subject can expect a clinical benefit from PD-1blockade treatment; an expression level of SMYD2 on CD8⁺ T cells in thePBMCs prior to PD-1 blockade greater than the reference level of SMYD2indicates that the subject can expect a clinical benefit from PD-1blockade treatment; an expression level of FBXO9 on CD8⁺ T cells in thePBMCs prior to PD-1 blockade greater than the reference level of FBXO9indicates that the subject can expect a clinical benefit from PD-1blockade treatment; an expression level of NCRNA00153 on CD8⁺ T cells inthe PBMCs prior to PD-1 blockade greater than the reference level ofNCRNA00153 indicates that the subject can expect a clinical benefit fromPD-1 blockade treatment; an expression level of NSMAF on CD8⁺ T cells inthe PBMCs prior to PD-1 blockade greater than the reference level ofNSMAF indicates that the subject can expect a clinical benefit from PD-1blockade treatment; an expression level of P2RY4 on CD8⁺ T cells in thePBMCs prior to PD-1 blockade lower than the reference level of P2RY4indicates that the subject can expect a clinical benefit from PD-1blockade treatment; an expression level of RCOR2 on CD8⁺ T cells in thePBMCs prior to PD-1 blockade lower than the reference level of RCOR2indicates that the subject can expect a clinical benefit from PD-1blockade treatment; an expression level of NUB1 on CD8⁺ T cells in thePBMCs prior to PD-1 blockade lower than the reference level of NUB1indicates that the subject can expect a clinical benefit from PD-1blockade treatment; an expression level of FCRL2 on CD8⁺ T cells in thePBMCs prior to PD-1 blockade lower than the reference level of FCRL2indicates that the subject can expect a clinical benefit from PD-1blockade treatment; an expression level of NUCKS1 on CD8⁺ T cells in thePBMCs prior to PD-1 blockade lower than the reference level of NUCKS1indicates that the subject can expect a clinical benefit from PD-1blockade treatment; an expression level of SHC2 on CD8⁺ T cells in thePBMCs prior to PD-1 blockade lower than the reference level of SHC2indicates that the subject can expect a clinical benefit from PD-1blockade treatment; an expression level of SCD5 on CD8⁺ T cells in thePBMCs prior to PD-1 blockade lower than the reference level of SCD5indicates that the subject can expect a clinical benefit from PD-1blockade treatment; an expression level of USP9Y on CD8⁺ T cells in thePBMCs prior to PD-1 blockade greater than the reference level of USP9Yindicates that the subject can expect a clinical benefit from PD-1blockade treatment; an expression level of HSPD1 on CD8⁺ T cells in thePBMCs prior to PD-1 blockade greater than the reference level of HSPD1indicates that the subject can expect a clinical benefit from PD-1blockade treatment; an expression level of CISH on CD8⁺ T cells in thePBMCs prior to PD-1 blockade greater than the reference level of CISHindicates that the subject can expect a clinical benefit from PD-1blockade treatment; an expression level of KLF12 on CD8⁺ T cells in thePBMCs prior to PD-1 blockade greater than the reference level of KLF12indicates that the subject can expect a clinical benefit from PD-1blockade treatment; an expression level of NCF4 on CD8⁺ T cells in thePBMCs prior to PD-1 blockade greater than the reference level of NCF4indicates that the subject can expect a clinical benefit from PD-1blockade treatment; an expression level of SFRS7 on CD8⁺ T cells in thePBMCs prior to PD-1 blockade greater than the reference level of SFRS7indicates that the subject can expect a clinical benefit from PD-1blockade treatment; an expression level of CDC14A on CD8⁺ T cells in thePBMCs prior to PD-1 blockade greater than the reference level of CDC14Aindicates that the subject can expect a clinical benefit from PD-1blockade treatment; an expression level of MAP2K5 on CD8⁺ T cells in thePBMCs prior to PD-1 blockade greater than the reference level of MAP2K5indicates that the subject can expect a clinical benefit from PD-1blockade treatment; an expression level of IL11RA on CD8⁺ T cells in thePBMCs prior to PD-1 blockade greater than the reference level of IL11RAindicates that the subject can expect a clinical benefit from PD-1blockade treatment; an expression level of MLL on CD8⁺ T cells in thePBMCs prior to PD-1 blockade greater than the reference level of MLLindicates that the subject can expect a clinical benefit from PD-1blockade treatment; an expression level of HSPBAP1 on CD8⁺ T cells inthe PBMCs prior to PD-1 blockade greater than the reference level ofHSPBAP1 indicates that the subject can expect a clinical benefit fromPD-1 blockade treatment; an expression level of MAP4K4 on CD8⁺ T cellsin the PBMCs prior to PD-1 blockade greater than the reference level ofMAP4K4 indicates that the subject can expect a clinical benefit fromPD-1 blockade treatment; an expression level of HELLS on CD8⁺ T cells inthe PBMCs prior to PD-1 blockade greater than the reference level ofHELLS indicates that the subject can expect a clinical benefit from PD-1blockade treatment; an expression level of C1QTNF3 on CD8⁺ T cells inthe PBMCs prior to PD-1 blockade greater than the reference level ofC1QTNF3 indicates that the subject can expect a clinical benefit fromPD-1 blockade treatment; an expression level of UBE2D1 on CD8⁺ T cellsin the PBMCs prior to PD-1 blockade greater than the reference level ofUBE2D1 indicates that the subject can expect a clinical benefit fromPD-1 blockade treatment; an expression level of FAIM3 on CD8⁺ T cells inthe PBMCs prior to PD-1 blockade greater than the reference level ofFAIM3 indicates that the subject can expect a clinical benefit from PD-1blockade treatment; an expression level of IFI44 on CD8⁺ T cells in thePBMCs prior to PD-1 blockade greater than the reference level of IFI44indicates that the subject can expect a clinical benefit from PD-1blockade treatment; an expression level of H2BFM on CD8⁺ T cells in thePBMCs prior to PD-1 blockade greater than the reference level of H2BFMindicates that the subject can expect a clinical benefit from PD-1blockade treatment; an expression level of GATAD1 on CD8⁺ T cells in thePBMCs prior to PD-1 blockade greater than the reference level of GATAD1indicates that the subject can expect a clinical benefit from PD-1blockade treatment; an expression level of CASP8 on CD8⁺ T cells in thePBMCs prior to PD-1 blockade greater than the reference level of CASP8indicates that the subject can expect a clinical benefit from PD-1blockade treatment; an expression level of SOCS1 on CD8⁺ T cells in thePBMCs prior to PD-1 blockade lower than the reference level of SOCS1indicates that the subject can expect a clinical benefit from PD-1blockade treatment; and an expression level of PDCD6 on CD8⁺ T cells inthe PBMCs prior to PD-1 blockade lower than the reference level of PDCD6indicates that the subject can expect a clinical benefit from PD-1blockade treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, and 1C are Kaplan-Meier relapse-free survival curvesshowing the proportion of patients with high baseline EOMES⁺/CD8⁺ (FIG.1A, dashed line), low EOMES⁺/CD8⁺ (FIG. 1A, solid line), high baselineKi67⁺EOMES⁺/CD8⁺ (FIG. 1B, dashed line), low Ki67⁺EOMES⁺/CD8⁺ (FIG. 1B,solid line), high level of CCR7 on CD4⁺ cells (FIG. 1C, dashed line), orlow level of CCR7 on CD4⁺ cells (FIG. 1C, solid line) as a function ofrelapse-free survival (months).

FIG. 2 is a graph of the percent of Ki67⁺EOMES⁺/CD8⁺ cells in CD8^(|)PBMCs for normal, relapsed, and NED subjects (subjects formerlydiagnosed with cancer but now showing no evidence of disease). p-valuesin the graph are from Wilcoxon rank-sum test. Overall p-value=0.038 forcomparing the three groups from Kruskal-Wallis test.

FIG. 3 is a Venn diagram of gene profiling impacted differentially byPD-1 blockade according to dose.

FIG. 4 is a principle component analysis (PCA) of pre and post treatmentgene expression of CD8⁺ T cells for separate clinical responders and nonresponders to PD-1 blockade treatment.

FIG. 5 is a heat map of baseline gene expression associated withclinical response by PD-1 blockade.

FIG. 6 is a heat map of post-treatment gene expression associated withclinical response by PD-1 blockade.

FIGS. 7A, 7B, 7C, and 7D are graphs of marker differences in respondersand nonresponders to treatment with BMS-936558. There were a total 25patients with 12 weeks measurement after BMS-936558 treatment; 13responders and 12 non-responders. FIG. 7A is a graph of the percent ofTregs in CD4⁺ T cells measured in responders and nonresponders pre andpost treatment with BMS-936558. FIG. 7B is a graph of the percent changein Tregs for responders (Yes) and nonresponders (No). Tregs (phenotypeas FoxP3+CD127LowCD25+CD4+) decreased in responders and increased innon-responders significantly. The change between two groups issignificant, too. FIG. 7C is a graph of the percent change inCD4^(|)/CLTA-4^(|) T cells for responders (Yes) and nonresponders (No).FIG. 7D is a graph of the percent change in CD8⁺/CLTA-4⁺ T cells forresponders (Yes) and nonresponders (No). CTLA-4+ in CD4 and CD8+ hassignificant change between responders and non-responders.

FIGS. 8A, 8B, 8C, and 8D are graphs of marker differences in respondersand nonresponders to treatment with BMS-936558. 33 patients weremeasured with baseline of the tetramers; 25 with 12 weeks measurement;13 responders and 12 non-responders. 8 patients only have baseline whoare non-responders. FIG. 8A is a graph of the percent of NY-ESO-1Tetramer^(|)CD8^(|) T cells measured in responders and nonrespondersbefore treatment with BMS-936558. FIG. 8B is a graph of the percent ofMART-1 Tetramer⁺ CD8⁺ T cells measured in responders and nonrespondersbefore treatment with BMS-936558. Both NY-ESO-1 and MART-1 specific CD8⁺T cells had higher baseline. Level in non-responders was compared toresponders. FIG. 8C is a graph of the percent of NY-ESO-1 Tetramer⁺ CD8⁺T cells measured in responders and nonresponders after treatment withBMS-936558. FIG. 8D is a graph of the percent of MART-1 Tetramer⁺ CD8⁺ Tcells measured in responders and nonresponders after treatment withBMS-936558. Responders had significant increase in both NY-ESO-1 andMART-1 specific CD8⁺ T cells at 12 weeks after BMS-936558 treatment.

FIGS. 9A and 9B are Box plots of CD71 baseline expression in irAE groupand non-irAE group of the patients. The line inside the rectangleindicated the median of the baseline CD71 expression distribution. Theupper and lower boundaries of the rectangle indicated the upper quartileand the lower quartile, respectively. Two lines (the whiskers) are drawnfrom the rectangle to the extreme values (highest and lowest expressionlevel of baseline CD71 expression in CD4+ T cells).

FIG. 10 is a graph of Kaplan-Meier curves for association of baselinebiomarkers (percentage of CD8⁺ T cells that are CD109⁺) with relapsefree survival.

FIG. 11 is a list of identified baseline biomarkers in CD4+ T cellsassociated with outcome (Relapse vs. NED).

FIG. 12 is a list of identified baseline biomarkers in CD8+ T cellsassociated with outcome (Relapse vs. NED).

FIG. 13 is a list of identified baseline biomarkers in CD4+ T cellsassociated with the induction of irAE (Yes vs. No).

FIG. 14 is a list of identified baseline biomarkers in CD8⁺ T cellsassociated with the induction of irAE (Yes vs. No).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The term “individual,” “host,” “subject,” and “patient” are usedinterchangeably to refer to any individual who is the target ofadministration or treatment. The subject can be a vertebrate, forexample, a mammal. Thus, the subject can be a human or veterinarypatient.

The term “treatment” refers to the medical management of a patient withthe intent to cure, ameliorate, stabilize, or prevent a disease,pathological condition, or disorder. This term includes activetreatment, that is, treatment directed specifically toward theimprovement of a disease, pathological condition, or disorder, and alsoincludes causal treatment, that is, treatment directed toward removal ofthe cause of the associated disease, pathological condition, ordisorder. In addition, this term includes palliative treatment, that is,treatment designed for the relief of symptoms rather than the curing ofthe disease, pathological condition, or disorder; preventativetreatment, that is, treatment directed to minimizing or partially orcompletely inhibiting the development of the associated disease,pathological condition, or disorder, and supportive treatment, that is,treatment employed to supplement another specific therapy directedtoward the improvement of the associated disease, pathologicalcondition, or disorder.

The term “cancer” or “malignant neoplasm” refers to a cell that displaysuncontrolled growth, invasion upon adjacent tissues, and oftenmetastasis to other locations of the body.

The term “metastasis” refers to the spread of malignant tumor cells fromone organ or part to another non-adjacent organ or part. Cancer cellscan “break away,” “leak,” or “spill” from a primary tumor, enterlymphatic and blood vessels, circulate through the bloodstream, andsettle down to grow within normal tissues elsewhere in the body. Whentumor cells metastasize, the new tumor is called a secondary ormetastatic cancer or tumor.

The term “biomarker” as used herein refers to a peptide, protein, ornucleic acid in or on a cell of a subject whose presence and/or level isindicative of a biological process, pathogenic process, or pharmacologicresponse to therapeutic intervention. In preferred embodiments,detection of the biomarker may be used to predict therapeutic outcome.

The term “relapse” as used herein refers to the return of a cancer aftera period of improvement, e.g., remission.

The term “immune related adverse event” or “irAE” refers to side effectsfrom CTLA-4 blockade therapy that are inflammatory in nature, includingrash, colitis, and hepatitis.

The term “frequency” as used herein and in the context of the disclosedmethods refers to the percentage of PBMCs expressing a particularbiomarker or combination of biomarkers at threshold levels. These levelsmay be determined empirically using standards and controls.

The term “expression level” as used herein refers to quantitative orrelative amounts of biomarker in or on a cell that is detected by anassay.

The level of expression measured for the disclosed biomarkers can becompared to any suitable control or standard level of expression. Forexample, a normal level (such as the level of the biomarker in a subjector subjects without cancer), a control level (such as the level of thebiomarker in a subject or subjects that do not respond and/or do not getclinical benefit from PD-1 blockade treatment), etc. can be used as acomparison to measured levels of expression of the biomarkers. Suchlevels used for comparison to measured expression levels of thebiomarkers can be referred to as reference levels.

The term “response,” in the context of CTLA-4 and PD-1 blockadetreatment, refers to a positive clinical response to the treatment.Related to this, the term “responder,” in the context of CTLA-4 and PD-1blockade treatment, refers to a subject that exhibits a response to thetreatment. Similarly, the term “non-response,” in the context of CTLA-4and PD-1 blockade treatment, refers to a lack of clinical response or anegative clinical response to the treatment; and the term“non-responder,” in the context of CTLA-4 and PD-1 blockade treatment,refers to a subject that exhibits a non-response to the treatment.

II. Biomarkers

A. CD8

In some embodiments, the biomarker is cluster of differentiation 8(CD8). CD8 is a transmembrane glycoprotein that serves as a co-receptorfor the T cell receptor (TCR). Like the TCR, CD8 binds to a majorhistocompatibility complex (MHC) molecule, but is specific for the classI MHC protein. There are two isoforms of the protein, alpha and beta,each encoded by a different gene. In humans, both genes are located onchromosome 2 in position 2p12. The CD8 co-receptor is predominantlyexpressed on the surface of cytotoxic T cells, but can also be found onnatural killer cells, cortical thymocytes, and dendritic cells. It isexpressed in T cell lymphoblastic lymphoma and hypo-pigmented mycosisfungoides, but is frequently lost in other T-cell neoplasms.

Antibodies that specifically bind human CD8 are commercially availableand include clone 3B5 (Life Technologies), clone SK1 (StemcellTechnologies), clone SP16 (Spring Biosciences), and OKT8 (eBioscience).

B. CD4

Cluster of differentiation 4 (CD4) is a glycoprotein expressed on thesurface of T helper cells, monocytes, macrophages, and dendritic cells.CD4 is a co-receptor that assists the T cell receptor (TCR) with anantigen-presenting cell. Using its portion that resides inside the Tcell, CD4 amplifies the signal generated by the TCR by recruiting anenzyme, known as the tyrosine kinase lck, which is essential foractivating many molecules involved in the signaling cascade of anactivated T cell. CD4 also interacts directly with MHC class IImolecules on the surface of the antigen-presenting cell using itsextracellular domain.

CD4 continues to be expressed in most neoplasms derived from T helpercells. It is therefore possible to use CD4 immunohistochemistry ontissue biopsy samples to identify most forms of peripheral T celllymphoma and related malignant conditions. The antigen has also beenassociated with a number of autoimmune diseases such as vitiligo andtype I diabetes mellitus.

Antibodies that specifically bind human CD4 are commercially availableand include clone L120 (Stemcell Technologies), clone SK3 (StemcellTechnologies), clone RPA-T4 (Imgenex), clone Q4120 (Sigma-Aldrich), andclone SP35 (Spring Biosciences).

C. EOMES

Eomesodermin homolog (EOMES) is a key transcription factor of cytotoxiclymphocyte lineages. Antibodies that specifically bind human EOMES arecommercially available and include clone 1A8 (Abnova), clone 2C7(Abnova), and clone 3E7 (MPBio). The nucleic acid sequence for humanEOMES is set forth in Accession No. NP_005433.

D. K167

The Ki-67 protein (also known as MKI67) is nuclear protein that isassociated with and may be necessary for cellular proliferation. Duringinterphase, Ki-67 can be exclusively detected within the cell nucleus,whereas in mitosis most of the protein is relocated to the surface ofthe chromosomes. Ki-67 protein is present during all active phases ofthe cell cycle (G1, S, G2, and mitosis), but is absent from restingcells (G0).

Ki-67 is an excellent marker to determine the growth fraction of a givencell population. The fraction of Ki-67-positive tumor cells (the Ki-67labeling index) is often correlated with the clinical course of cancer.The best-studied examples in this context are carcinomas of theprostate, brain and the breast. For these types of tumors, theprognostic value for survival and tumor recurrence have repeatedly beenproven in uni- and multivariate analysis.

MIB1 is a commonly used monoclonal antibody that detects the Ki-67antigen. It is used in clinical applications to determine the Ki-67labeling index. One of its primary advantages over the original Ki-67antibody (and the reason why it has essentially supplanted the originalantibody for clinical use) is that it can be used on formalin-fixedparaffin-embedded sections, after heat-mediated antigen retrieval (seesection Original Ki-67 antibody).

E. TGFβR3

TGFβ receptors are single pass serine/threonine kinase receptors. Theyexist in several different isoforms that can be homo- or heterodimeric.The number of characterized ligands in the TGFβ superfamily far exceedsthe number of known receptors, suggesting the promiscuity that existsbetween the ligand and receptor interactions. Three TGF-β receptor typescan be distinguished by their structural and functional properties.Receptor types I and II have similar ligand-binding affinities and canbe distinguished from each other only by peptide mapping. Both receptortypes I and II have a high affinity for TGF-01 and low affinity forTGF-β2. TGF-β receptor type III (TGFβR3) has a high affinity for bothTGF-β1 and TGF-32.

Antibodies that specifically bind human TGFβR3 are commerciallyavailable and include clone ZT001 (Invitrogen), clone D11G10 (CellSignaling Technologies), and clone 20724 (R&D Systems). The nucleic acidsequence for human TGFβR3 is set forth in Accession No. NM_003243.

F. CCR7

C—C chemokine receptor type 7 (CCR7) is expressed in various lymphoidtissues and activates B and T lymphocytes. It has been shown to controlthe migration of memory T cells home to lymph nodes, as well asstimulate dendritic cell maturation. CCR7 signaling is also known toplay a role in lymph node metastasis. The chemokine (C—C motif) ligand19 (CCL 19/ELC) has been reported to be a specific ligand of thisreceptor.

Antibodies that specifically bind human CCR7 are commercially availableand include clone 150503 (R&D Systems), clone FR11-11E8 (MiltenyiBiotec), and clone 3D12 (eBioscience). The nucleic acid sequence forhuman CCR7 is set forth in Accession No. NM_001838.2.

G. CD71

CD71, also known as transferrin receptor protein 1 (TfR1), is a proteinencoded by the TFRC gene. CD71 is required for iron delivery fromtransferrin to cells.

Antibodies that specifically bind human CD71 are commercially availableand include GR08K-100UG, GR09L-100UG, CBL47 (available from EMDMillipore); and clone 29806 (available from R&D Systems). The nucleicacid sequence for human CD71 is set forth in Accession No. NM_001128148.

H. CD109

CD109 is a GPI-linked cell surface antigen expressed by CD34+ acutemyeloid leukemia cell lines, T-cell lines, activated T lymphoblasts,endothelial cells, and activated platelets (Lin et al., Blood 99(5):1683-91 (2002)). CD109 binds to and negatively regulates signaling oftransforming growth factor beta (TGF-β).

Antibodies that specifically bind human ICOS are commercially availableand include clone 496920 (available from R&D Systems) and cloneNBP1-84393 (available from Novus Biologicals). The nucleic acid sequencefor human CD109 is set forth in Accession No. NM_001159587.

I. MART-1

Melanoma antigen recognized by T-cells 1 (MART-1), also known asmelan-A, is encoded by the MLANA gene. A fragment of the protein,usually consisting of the nine amino acids 27 to 35, is bound by MHCclass I complexes which present it to T cells of the immune system.These complexes can be found on the surface of melanoma cells. Decamericpeptides (26-35) are being investigated as cancer vaccines.

Antibodies that specifically bind human MART-1 are commerciallyavailable and include clone EP1422Y (available from Novus Biologicals)and clone A103 (available from EMD Millipore). The nucleic acid sequencefor human MART-1 is set forth in Accession No. NM_005511.

J. NY-ESO-1

Cancer/testis antigen 1 (NY-ESO-1) is a protein expressed in testis andvarious cancers. Antibodies that specifically bind human NY-ESO-1 arecommercially available and include clone 3F10 (available from OriGene)and clone 2B6 (available from Novus Biologicals). The nucleic acidsequence for human NY-ESO-1 is set forth in Accession No. NM_001327.

III. Methods

Methods are disclosed for predicting the efficacy, potential forrelapse, and irAE in a subject diagnosed with cancer and undergoingtreatment. Methods are also disclosed for predicting the efficacy,potential for relapse, and irAE in a subject diagnosed with cancer andundergoing treatment based on the levels of specific biomarkers measuredin the subject. Methods are also disclosed for treating subjectsdiagnosed with cancer based on the predicted efficacy, potential forrelapse, and irAE in the subject. Methods are also disclosed fortreating specific subjects selected from subjects diagnosed with cancerbased on the predicted efficacy, potential for relapse, and irAE in thesubject. Methods are also disclosed for treating subjects diagnosed withcancer based on the levels of specific biomarkers measured in thesubject. Methods are also disclosed for treating specific subjectsselected from subjects diagnosed with cancer based on the levels ofspecific biomarkers measured in the subject.

Methods are also disclosed for not treating subjects diagnosed withcancer with specific therapies based on the predicted efficacy,potential for relapse, and irAE in the subject. Methods are alsodisclosed for not treating specific subjects selected from subjectsdiagnosed with cancer with specific therapies based on the predictedefficacy, potential for relapse, and irAE in the subject. Methods arealso disclosed for not treating subjects diagnosed with cancer withspecific therapies based on the levels of specific biomarkers measuredin the subject. Methods are also disclosed for not treating specificsubjects selected from subjects diagnosed with cancer with specifictherapies based on the levels of specific biomarkers measured in thesubject.

Methods are disclosed for predicting the efficacy and clinical benefitin a subject diagnosed with cancer and undergoing treatment. Methods arealso disclosed for predicting the efficacy and clinical benefit in asubject diagnosed with cancer and undergoing treatment based on thelevels of specific biomarkers measured in the subject. Methods are alsodisclosed for treating subjects diagnosed with cancer based on thepredicted efficacy and clinical benefit in the subject. Methods are alsodisclosed for treating specific subjects selected from subjectsdiagnosed with cancer based on the predicted efficacy and clinicalbenefit in the subject. Methods are also disclosed for treating subjectsdiagnosed with cancer based on the levels of specific biomarkersmeasured in the subject. Methods are also disclosed for treatingspecific subjects selected from subjects diagnosed with cancer based onthe levels of specific biomarkers measured in the subject.

Methods are also disclosed for not treating subjects diagnosed withcancer with specific therapies based on the predicted efficacy andclinical benefit in the subject. Methods are also disclosed for nottreating specific subjects selected from subjects diagnosed with cancerwith specific therapies based on the predicted efficacy and clinicalbenefit in the subject. Methods are also disclosed for not treatingsubjects diagnosed with cancer with specific therapies based on thelevels of specific biomarkers measured in the subject. Methods are alsodisclosed for not treating specific subjects selected from subjectsdiagnosed with cancer with specific therapies based on the levels ofspecific biomarkers measured in the subject.

The cancer of the disclosed methods can be any cell in a subjectundergoing unregulated growth, invasion, or metastasis. In some aspects,the cancer can be any neoplasm or tumor for which radiotherapy iscurrently used. Alternatively, the cancer can be a neoplasm or tumorthat is not sufficiently sensitive to radiotherapy using standardmethods. Thus, the cancer can be a sarcoma, lymphoma, leukemia,carcinoma, blastoma, or germ cell tumor. A representative butnon-limiting list of cancers that the disclosed compositions can be usedto treat include lymphoma, B cell lymphoma, T cell lymphoma, mycosisfungoides, Hodgkin's Disease, myeloid leukemia, bladder cancer, braincancer, nervous system cancer, head and neck cancer, squamous cellcarcinoma of head and neck, kidney cancer, lung cancers such as smallcell lung cancer and non-small cell lung cancer,neuroblastoma/glioblastoma, ovarian cancer, pancreatic cancer, prostatecancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas ofthe mouth, throat, larynx, and lung, colon cancer, ccrvical cancer,ccrvical carcinoma, breast cancer, epithelial cancer, renal cancer,genitourinary cancer, pulmonary cancer, esophageal carcinoma, head andneck carcinoma, large bowel cancer, hematopoietic cancers; testicularcancer; colon and rectal cancers, prostatic cancer, and pancreaticcancer.

In preferred embodiments, the cancer is a melanoma. In theseembodiments, the method may be used to predict efficacy of CTLA-4blockade therapy, treatment with other neoplastic drugs, or treatmentwith a combination of CTLA-4 blockade therapy and other neoplasticdrugs.

In preferred embodiments, the cancer is a melanoma. In theseembodiments, the method may be used to predict efficacy and clinicalbenefit of PD-1 blockade therapy, treatment with other neoplastic drugs,or treatment with a combination of PD-1 blockade therapy and otherneoplastic drugs.

The disclosed methods involve detecting one or more biomarkers in or oncells in the subject. In preferred embodiments, the one or morebiomarkers can be detected in or on PBMCs in the subject, T cells in thesubject, T cells in the subject's PBMCs, CD8⁺ T cells in the subject,CD4⁺ T cells in the subject, CD8⁺ T cells in the subject's PBMCs, CD4⁺ Tcells in the subject's PBMCs, or a combination. In most preferredembodiments the one or more biomarkers can be detected in or on PBMCs inthe subject. Biomarkers can be detected using standard methods, such asimmunoassays to detect proteins in or on the cells, or nucleic aciddetection methods to detect gene expression within the cells.

A. Immunoassays

In some aspects, one or more biomarkers are detected using animmunoassay. Many types and formats of immunoassays are known and allare suitable for detecting the disclosed biomarkers. Examples ofimmunoassays are enzyme linked immunosorbent assays (ELISAs),radioimmunoassays (RIA), radioimmune precipitation assays (RIPA),immunobead capture assays, Western blotting, dot blotting, gel-shiftassays, flow cytometry, protein arrays, multiplexed bead arrays,magnetic capture, in vivo imaging, fluorescence resonance energytransfer (FRET), and fluorescence rccovcry/localization aftcrphotoblcaching (FRAP/FLAP).

In general, immunoassays involve contacting a sample suspected ofcontaining a molecule of interest (such as the disclosed biomarkers)with an antibody to the molecule of interest or contacting an antibodyto a molecule of interest (such as antibodies to the disclosedbiomarkers) with a molecule that can be bound by the antibody, as thecase may be, under conditions effective to allow the formation ofimmunocomplexes. Contacting a sample with the antibody to the moleculeof interest or with the molecule that can be bound by an antibody to themolecule of interest under conditions effective and for a period of timesufficient to allow the formation of immune complexes (primary immunecomplexes) is generally a matter of simply bringing into contact themolecule or antibody and the sample and incubating the mixture for aperiod of time long enough for the antibodies to form immune complexeswith, i.e., to bind to, any molecules (e.g., antigens) present to whichthe antibodies can bind. In many forms of immunoassay, thesample-antibody composition, such as a tissue section, ELISA plate, dotblot or Western blot, can then be washed to remove any non-specificallybound antibody species, allowing only those antibodies specificallybound within the primary immune complexes to be detected.

Immunoassays can include methods for detecting or quantifying the amountof a molecule of interest (such as the disclosed biomarkers or theirantibodies) in a sample, which methods generally involve the detectionor quantitation of any immune complexes formed during the bindingprocess. In general, the detection of immunocomplex formation is wellknown in the art and can be achieved through the application of numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any radioactive, fluorescent, biological orenzymatic tags or any other known label.

As used herein, a label can include a fluorescent dye, a member of abinding pair, such as biotin/streptavidin, a metal (e.g., gold), or anepitope tag that can specifically interact with a molecule that can bedetected, such as by producing a colored substrate or fluorescence.Substances suitable for detectably labeling proteins include fluorescentdyes (also known herein as fluorochromes and fluorophores) and enzymesthat react with colorometric substrates (e.g., horseradish peroxidase).The use of fluorescent dyes is generally preferred in the practice ofthe invention as they can be detected at very low amounts. Furthermore,in the case where multiple antigens are reacted with a single array,each antigen can be labeled with a distinct fluorescent compound forsimultaneous detection. Labeled spots on the array are detected using afluorimeter, the presence of a signal indicating an antigen bound to aspecific antibody.

A modifier unit such as a radionuclide can be incorporated into orattached directly to any of the compounds described herein byhalogenation. In another aspect, the radionuclide can be attached to alinking group or bound by a chelating group, which is then attached tothe compound directly or by means of a linker. Radiolabeling techniquessuch as these are routinely used in the radiopharmaceutical industry.

Labeling can be either direct or indirect. In direct labeling, thedetecting antibody (the antibody for the molecule of interest) ordetecting molecule (the molecule that can be bound by an antibody to themolecule of interest) include a label. Detection of the label indicatesthe presence of the detecting antibody or detecting molecule, which inturn indicates the presence of the molecule of interest or of anantibody to the molecule of interest, respectively. In indirectlabeling, an additional molecule or moiety is brought into contact with,or generated at the site of, the immunocomplex. For example, asignal-generating molecule or moiety such as an enzyme can be attachedto or associated with the detecting antibody or detecting molecule. Thesignal-generating molecule can then generate a detectable signal at thesite of the immunocomplex. For example, an enzyme, when supplied withsuitable substrate, can produce a visible or detectable product at thesite of the immunocomplex. ELISAs use this type of indirect labeling.

As another example of indirect labeling, an additional molecule (whichcan be referred to as a binding agent) that can bind to either themolecule of interest or to the antibody (primary antibody) to themolecule of interest, such as a second antibody to the primary antibody,can be contacted with the immunocomplex. The additional molecule canhave a label or signal-generating molecule or moiety. The additionalmolecule can be an antibody, which can thus be termed a secondaryantibody. Binding of a secondary antibody to the primary antibody canform a so-called sandwich with the first (or primary) antibody and themolecule of interest. The immune complexes can be contacted with thelabeled, secondary antibody under conditions effective and for a periodof time sufficient to allow the formation of secondary immune complexes.The secondary immune complexes can then be generally washed to removeany non-specifically bound labeled secondary antibodies, and theremaining label in the secondary immune complexes can then be detected.The additional molecule can also be or include one of a pair ofmolecules or moieties that can bind to each other, such as thebiotin/avadin pair. In this mode, the detecting antibody or detectingmolecule should include the other member of the pair.

Other modes of indirect labeling include the detection of primary immunecomplexes by a two-step approach. For example, a molecule (which can bereferred to as a first binding agent), such as an antibody, that hasbinding affinity for the molecule of interest or corresponding antibodycan be used to form secondary immune complexes, as described above.After washing, the secondary immune complexes can be contacted withanother molecule (which can be referred to as a second binding agent)that has binding affinity for the first binding agent, again underconditions effective and for a period of time sufficient to allow theformation of immune complexes (thus forming tertiary immune complexes).The second binding agent can be linked to a detectable label orsignal-generating molecule or moiety, allowing detection of the tertiaryimmune complexes thus formed. This system can provide for signalamplification.

Immunoassays that involve the detection of as substance, such as aprotein or an antibody to a specific protein, include label-free assays,protein separation methods (i.e., electrophoresis), solid supportcapture assays, or in vivo detection. Label-free assays are generallydiagnostic means of determining the presence or absence of a specificprotein, or an antibody to a specific protein, in a sample. Proteinseparation methods are additionally useful for evaluating physicalproperties of the protein, such as size or net charge. Capture assaysare generally more useful for quantitatively evaluating theconcentration of a specific protein, or antibody to a specific protein,in a sample. Finally, in vivo detection is useful for evaluating thespatial expression patterns of the substance, i.e., where the substancecan be found in a subject, tissue or cell.

Flow cytometry is a well-known technique for counting and/or otherwiseexamining microscopic particles, such as cells and the like, by passinga stream of fluid in which the particles are suspended through adetection apparatus. The detection apparatus typically relies ondetecting the optical response produced as the particles pass through anilluminated region of the device. In some forms, for example, individualparticles pass through an illumination zone, typically at a rate on theorder of 1,000 cells per second, and detectors, gated electronically,measure the magnitude of a pulse representing the light scattered by thecells. The pulse magnitudes (or other properties) may then be processedto characterize the cells by a particular parameter of interest. Forexample, the angular dependence of scattered light may provideinformation on the nature of the scattering particles. More importantly,the fluorescent properties of the particles (which may be caused byappropriate fluorophores being added to the suspension) may providedesired parametric information. Exemplary flow cytometry systems aredisclosed in U.S. Pat. No. 5,760,900 and U.S. Patent Publication No.2008/0186479, which are hereby incorporated by reference herein in theirentirety. Those of skill in the art are aware of other systems andtechniques for flow cytometry, any of which can be adapted for use withthe disclosed methods.

B. Nucleic Acid Detection

In some aspects, one or more biomarkers are nucleic acids, such as mRNA.A number of widely used procedures exist for detecting and determiningthe abundance of a particular mRNA in a total or poly(A) RNA sample. Forexample, specific mRNAs can be detected using Northern blot analysis,nuclease protection assays (NPA), in situ hybridization, or reversetranscription-polymerase chain reaction (RT-PCR).

In theory, each of these techniques can be used to detect specific RNAsand to precisely determine their expression level. In general, Northernanalysis is the only method that provides information about transcriptsize, whereas NPAs are the easiest way to simultaneously examinemultiple messages. In situ hybridization is used to localize expressionof a particular gene within a tissue or cell type, and RT-PCR is themost sensitive method for detecting and quantitating gene expression.

Northern analysis presents several advantages over the other techniques.The most compelling of these is that it is the easiest method fordetermining transcript size, and for identifying alternatively splicedtranscripts and multigene family members. It can also be used todirectly compare the relative abundance of a given message between allthe samples on a blot. The Northern blotting procedure isstraightforward and provides opportunities to evaluate progress atvarious points (e.g., intactness of the RNA sample and how efficientlyit has transferred to the membrane). RNA samples are first separated bysize via electrophoresis in an agarose gel under denaturing conditions.The RNA is then transferred to a membrane, crosslinked and hybridizedwith a labeled probe. Nonisotopic or high specific activity radiolabeledprobes can be used including random-primed, nick-translated, orPCR-generated DNA probes, in vitro transcribed RNA probes, andoligonucleotides. Additionally, sequences with only partial homology(e.g., cDNA from a different species or genomic DNA fragments that mightcontain an exon) may be used as probes.

The Nuclease Protection Assay (NPA) (including both ribonucleaseprotection assays and SI nuclease assays) is an extremely sensitivemethod for the detection and quantitation of specific mRNAs. The basisof the NPA is solution hybridization of an antisense probe (radiolabeledor nonisotopic) to an RNA sample. After hybridization, single-stranded,unhybridized probe and RNA are degraded by nucleases. The remainingprotected fragments are separated on an acrylamide gel. Solutionhybridization is typically more efficient than membrane-basedhybridization, and it can accommodate up to 100 μg of sample RNA,compared with the 20-30 μg maximum of blot hybridizations. NPAs are alsoless sensitive to RNA sample degradation than Northern analysis sincecleavage is only detected in the region of overlap with the probe(probes are usually about 100-400 bases in length). NPAs are the methodof choice for the simultaneous detection of several RNA species. Duringsolution hybridization and subsequent analysis, individual probe/targetinteractions are completely independent of one another. Thus, severalRNA targets and appropriate controls can be assayed simultaneously (upto twelve have been used in the same reaction), provided that theindividual probes are of different lengths. NPAs are also commonly usedto precisely map mRNA termini and intron/exon junctions.

In situ hybridization (ISH) is a powerful and versatile tool for thelocalization of specific mRNAs in cells or tissues. Unlike Northernanalysis and nuclease protection assays, ISH does not require theisolation or electrophoretic separation of RNA. Hybridization of theprobe takes place within the cell or tissue. Since cellular structure ismaintained throughout the procedure, ISH provides information about thelocation of mRNA within the tissue sample. The procedure begins byfixing samples in neutral-buffered formalin, and embedding the tissue inparaffin. The samples are then sliced into thin sections and mountedonto microscope slides. (Alternatively, tissue can be sectioned frozenand post-fixed in paraformaldehyde.) After a series of washes to dewaxand rehydrate the sections, a Proteinase K digestion is performed toincrease probe accessibility, and a labeled probe is then hybridized tothe sample sections. Radiolabeled probes are visualized with liquid filmdried onto the slides, while nonisotopically labeled probes areconveniently detected with colorimetric or fluorescent reagents.

RT-PCR has revolutionized the study of gene expression. It is nowtheoretically possible to detect the RNA transcript of any gene,regardless of the scarcity of the starting material or relativeabundance of the specific mRNA. In RT-PCR, an RNA template is copiedinto a complementary DNA (cDNA) using a retroviral reversetranscriptase. The cDNA is then amplified exponentially by PCR. As withNPAs, RT-PCR is somewhat tolerant of degraded RNA. As long as the RNA isintact within the region spanned by the primers, the target will beamplified.

Relative quantitative RT-PCR involves amplifying an internal controlsimultaneously with the gene of interest. The internal control is usedto normalize the samples. Once normalized, direct comparisons ofrelative abundance of a specific mRNA can be made across the samples. Itis crucial to choose an internal control with a constant level ofexpression across all experimental samples (i.e., not affected byexperimental treatment). Commonly used internal controls (e.g., GAPDH,tactin, cyclophilin) often vary in expression and, therefore, may not beappropriate internal controls. Additionally, most common internalcontrols are expressed at much higher levels than the mRNA beingstudied. For relative RT-PCR results to be meaningful, all products ofthe PCR reaction must be analyzed in the linear range of amplification.This becomes difficult for transcripts of widely different levels ofabundance.

Competitive RT-PCR is used for absolute quantitation. This techniqueinvolves designing, synthesizing, and accurately quantitating acompetitor RNA that can be distinguished from the endogenous target by asmall difference in size or sequence. Known amounts of the competitorRNA are added to experimental samples and RT-PCR is performed. Signalsfrom the endogenous target are compared with signals from the competitorto determine the amount of target present in the sample.

C. CTLA-4 Blockade Therapies

CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) is a molecule oncytotoxic T lymphocytes that is believed to play a critical role inregulating natural immune responses. The absence or presence of CTLA-4can augment or suppress the immune system's T-cell response in fightingdisease. Antibodies designed to block the activity of CTLA-4 areeffective in sustaining an active immune response in its attack oncancer cells. CTLA-4 blockade therapy can, for example, target CTLA-4.The composition or compound used can block the function of the CTLA-4.

For example, Ipilimumab is a fully human antibody that binds to CTLA-4.Ipilimumab was approved by the FDA in March 2011 to treat patients withlate-stage melanoma that has spread or cannot be removed by surgery.Additionally, ipilimumab is undergoing clinical trials for the treatmentof non-small cell lung carcinoma (NSCLC), small cell lung cancer (SCLC)and metastatic hormone-refractory prostate cancer. Tremelimumab is afully human IgG2 monoclonal antibody that binds to CTLA-4.

D. Non CTLA-4 Anti-Neoplastic Therapies

Numerous anti-cancer (antineoplastic) drugs are available for treatingsubjects with cancer. In some embodiments, the disclosed biomarkers mayalso be used to predict efficacy, relapse, and irAE in a subject treatedwith one or more of these anti-neoplastic drugs, alone or in combinationwith CTLA-4 blockade therapy.

E. PD-1 Blockade Therapies

Programmed Death-1 (PD-1) is a negative regulator of T cell activationand proliferation that mediates suppressive action by binding to itsligands PD-L1 and PD-L2. T cell exhaustion is a state of T celldysfunction that arises during many chronic infections and cancer. Overthe last few years, it has been established that PD-1 plays an activeand reversible role in T cell exhaustion. Under physiologicalconditions, PD-1 is induced after T cell activation and serves as aninhibitory feedback to dampen the TCR signaling cascade and preventexcessive T cell activation. PD-1 plays also an important role in thetolerance to self antigens. Anti-PD-1 antibodies, such as BMS-936558 andBMS-936559, have been identified as potential cancer therapeutic.BMS-936558 is an antibody against PD-1 and is currently in clinicaltrials. PD-1 blockade therapy can target either or both the PD-1receptor or the ligand of the PD-1 receptor. The composition or compoundused can block the function of the PD-1 receptor.

F. Non PD-1 Anti-Neoplastic Therapies

Numerous anti-cancer (antineoplastic) drugs are available for treatingsubjects with cancer. In some embodiments, the disclosed biomarkers mayalso be used to predict efficacy and clinical benefit in a subjecttreated with one or more of these anti-neoplastic drugs, alone or incombination with PD-1 blockade therapy.

Antineoplastic drugs include Acivicin, Aclarubicin, AcodazoleHydrochloride, AcrQnine, Adozelesin, Aldesleukin, Altretamine,Ambomycin, Ametantrone Acetate, Aminoglutethimide, Amsacrine,Anastrozole, Anthramycin, Asparaginase, Asperlin, Azacitidine, Azetepa,Azotomycin, Batimastat, Benzodepa, Bicalutamide, BisantreneHydrochloride, Bisnafide Dimesylate, Bizelesin, Bleomycin Sulfate,Brequinar Sodium, Bropirimine, Busulfan, Cactinomycin, Calusterone,Caraccmide, Carbctimer, Carboplatin, Carmustine, CarubicinHydrochloride, Carzelesin, Cedefingol, Chlorambucil, Cirolemycin,Cisplatin, Cladribine, Crisnatol Mesylate, Cyclophosphamide, Cytarabine,Dacarbazine, Dactinomycin, Daunorubicin Hydrochloride, Decitabine,Dexormaplatin, Dezaguanine, Dezaguanine Mesylate, Diaziquone, Docetaxel,Doxorubicin, Doxorubicin Hydrochloride, Droloxifene, DroloxifeneCitrate, Dromostanolone Propionate, Duazomycin, Edatrexate, EflomithineHydrochloride, Elsamitrucin, Enloplatin, Enpromate, Epipropidine,Epirubicin Hydrochloride, Erbulozole, Esorubicin Hydrochloride,Estramustine, Estramustine Phosphate Sodium, Etanidazole, Ethiodized OilI 131, Etoposide, Etoposide Phosphate, Etoprine, FadrozoleHydrochloride, Fazarabine, Fenretinide, Floxuridine, FludarabinePhosphate, Fluorouracil, Flurocitabine, Fosquidone, Fostriecin Sodium,Gemcitabine, Gemcitabine Hydrochloride, Gold Au 198, Hydroxyurea,Idarubicin Hydrochloride, Ifosfamide, Ilmofosine, Interferon Alfa-2a,Interferon Alfa-2b, Interferon Alfa-n1, Interferon Alfa-n3, InterferonBeta-Ia, Interferon Gamma-Ib, Iproplatin, Irinotecan Hydrochloride,Lanreotide Acetate, Letrozole, Leuprolide Acetate, LiarozoleHydrochloride, Lometrexol Sodium, Lomustine, Losoxantrone Hydrochloride,Masoprocol, Maytansine, Mechlorethamine Hydrochloride, MegestrolAcetate, Melengestrol Acetate, Melphalan, Menogaril, Mercaptopurine,Methotrexate, Methotrexate Sodium, Metoprine, Meturedepa, Mitindomide,Mitocarcin, Mitocromin, Mitogillin, Mitomalcin, Mitomycin, Mitosper,Mitotane, Mitoxantrone Hydrochloride, Mycophenolic Acid, Nocodazole,Nogalamycin, Ormaplatin, Oxisuran, Paclitaxel, Pegaspargase, Peliomycin,Pentamustine, Peplomycin Sulfate, Perfosfamide, Pipobroman, Piposulfan,Piroxantrone Hydrochloride, Plicamycin, Plomestane, Porfimer Sodium,Porfiromycin, Prednimustine, Procarbazine Hydrochloride, Puromycin,Puromycin Hydrochloride, Pyrazofurin, Riboprine, Rogletimide, Safmgol,Safingol Hydrochloride, Semustine, Simtrazene, Sparfosate Sodium,Sparsomycin, Spirogermanium Hydrochloride, Spiromustine, Spiroplatin,Streptonigrin, Streptozocin, Strontium Chloride Sr 89, Sulofenur,Talisomycin, Taxane, Taxoid, Tecogalan Sodium, Tegafur, TeloxantroneHydrochloride, Temoporfin, Teniposide, Teroxirone, Testolactone,Thiamiprine, Thioguanine, Thiotepa, Tiazofurin, Tirapazamine, TopotecanHydrochloride, Torcmifene Citrate, Trestolone Acetate, TriciribinePhosphate, Trimetrexate, Trimetrexate Glucuronate, Triptorelin,Tubulozole Hydrochloride, Uracil Mustard, Uredepa, Vapreotide,Verteporfin, Vinblastine Sulfate, Vincristine Sulfate, Vindesine,Vindesine Sulfate, Vinepidine Sulfate, Vinglycinate Sulfate,Vinleurosine Sulfate, Vinorelbine Tartrate, Vinrosidine Sulfate,Vinzolidine Sulfate, Vorozole, Zeniplatin, Zinostatin, ZorubicinHydrochloride.

G. Monitoring Treatment

Biomarkers have also been discovered that can be used to monitorsubjects following treatment to determine the effectiveness of thetreatment and/or the prognosis for the subject. For example, certainbiomarkers are associated with patients less likely to relapse or anirAE following CTLA-4 blockade therapy. As another example, certainbiomarkers are associated with patients more likely to respond totreatment following PD-1 blockade therapy.

A decrease of Ki67⁺EOMES⁺CD4⁺ cells in CD4⁺ T cells, a decrease inexpression of CCR7 on CD8⁺ T cells, a decrease in the frequency ofCCR7⁺CD8⁺, cells in CD8⁺ T cells, an increase in expression of TGFβR3 onCD8⁺ T cells, an increase in expression of EOMES in CD8⁺ cells, anincrease in frequency of EOMES⁺CD8⁺ cells in CD8⁺ T cells, an increasein the frequency of GranzymeB⁺EOMES⁺CD8⁺ cells in CD8⁺ T cells, anincrease in the frequency of Perforin⁺EOMES⁺CD8⁺ cells in CD8⁺ T cells,an increase in the frequency of CTLA-4⁺CD4⁺ cells in CD4⁺ T cells, or acombination indicates a higher risk of relapse (that is, a poorerprognosis or an indication that therapy is not successful) in subjectswith cancer that have been treated with CTLA-4 blockade therapy. Adecrease of Ki67⁺CD8⁺ cells in CD8⁺ T cells, a decrease of CD71⁺CD8⁺cells in CD8⁺ T cells, a decrease of ICOS⁺CD8⁺ cells in CD8⁺ T cells, adecrease of PD-1⁺CD8⁺ cells in CD8⁺ T cells, or a combination indicatesa higher risk of developing irAE (that is, a poorer prognosis or anindication that therapy is not successful) in subjects with cancer thathave been treated with CTLA-4 blockade therapy. Thus, these biomarkerscan be used to assess the progress and effectiveness of CTLA-4 blockadetreatment.

An increase in NY-ESO-1⁺CD8⁺ cells in CD8⁺ T cells or an increase inMART-1^(|)CD8^(|) cells in CD8+ T cells, or a combination indicates alower risk of relapse (that is, a better prognosis or an indication thattherapy is successful) in subjects with cancer that have been treatedwith PD-1 blockade therapy (see FIGS. 8C and 8D).

The disclosed methods include the determination, identification,indication, correlation, diagnosis, prognosis, etc. (which can bereferred to collectively as “identifications”) of subjects, diseases,conditions, states, etc. based on measurements, detections, comparisons,analyses, assays, screenings, etc. For example, subjects diagnosed withcancer can be identified for specific treatments (or avoidance ofspecific treatments) based on the levels of specific biomarkers measuredin the subject Such identifications are useful for many reasons. Forexample, and in particular, such identifications allow specific actionsto be taken based on, and relevant to, the particular identificationmade. For example, diagnosis of a particular disease or condition inparticular subjects (and the lack of diagnosis of that disease orcondition in other subjects) has the very useful effect of identifyingsubjects that would benefit from treatment, actions, behaviors, etc.based on the diagnosis. For example, treatment for a particular diseaseor condition in subjects identified is significantly different fromtreatment of all subjects without making such an identification (orwithout regard to the identification). Subjects needing or that couldbenefit from the treatment will receive it and subjects that do not needor would not benefit from the treatment will not receive it.

Accordingly, also disclosed herein are methods comprising takingparticular actions following and based on the disclosed identifications.For example, disclosed are methods comprising creating a record of anidentification (in physical—such as paper, electronic, or other-form,for example). Thus, for example, creating a record of an identificationbased on the disclosed methods differs physically and tangibly frommerely performing a measurement, detection, comparison, analysis, assay,screen, etc. Such a record is particularly substantial and significantin that it allows the identification to be fixed in a tangible form thatcan be, for example, communicated to others (such as those who couldtreat, monitor, follow-up, advise, etc. the subject based on theidentification); retained for later use or review; used as data toassess sets of subjects, treatment efficacy, accuracy of identificationsbased on different measurements, detections, comparisons, analyses,assays, screenings, etc., and the like. For example, such uses ofrecords of identifications can be made, for example, by the sameindividual or entity as, by a different individual or entity than, or acombination of the same individual or entity as and a differentindividual or entity than, the individual or entity that made the recordof the identification. The disclosed methods of creating a record can becombined with any one or more other methods disclosed herein, and inparticular, with any one or more steps of the disclosed methods ofidentification.

As another example, disclosed are methods comprising making one or morefurther identifications based on one or more other identifications. Forexample, particular treatments, monitorings, follow-ups, advice, etc.can be identified based on the other identification. For example,identification of a subject as having a disease or condition with a highlevel of a particular component or characteristic can be furtheridentified as a subject that could or should be treated with a therapybased on or directed to the high level component or characteristic. Arecord of such further identifications can be created (as describedabove, for example) and can be used in any suitable way. Such furtheridentifications can be based, for example, directly on the otheridentifications, a record of such other identifications, or acombination. Such further identifications can be made, for example, bythe same individual or entity as, by a different individual or entitythan, or a combination of the same individual or entity as and adifferent individual or entity than, the individual or entity that madethe other identifications. The disclosed methods of making a furtheridentification can be combined with any one or more other methodsdisclosed herein, and in particular, with any one or more steps of thedisclosed methods of identification.

As another example, disclosed are methods comprising treating,monitoring, following-up with, advising, etc., a subject identified inany of the disclosed methods. Also disclosed are methods comprisingtreating, monitoring, following-up with, advising, etc., a subject forwhich a record of an identification from any of the disclosed methodshas been made. For example, particular treatments, monitorings,follow-ups, advice, etc. can be used based on an identification and/orbased on a record of an identification. For example, a subjectidentified as having a disease or condition with a high level of aparticular component or characteristic (and/or a subject for which arecord has been made of such an identification) can be treated with atherapy based on or directed to the high level component orcharacteristic. Such treatments, monitorings, follow-ups, advice, etc.can be based, for example, directly on identifications, a record of suchidentifications, or a combination. Such treatments, monitorings,follow-ups, advice, etc. can be performed, for example, by the sameindividual or entity as, by a different individual or entity than, or acombination of the same individual or entity as and a differentindividual or entity than, the individual or entity that made theidentifications and/or record of the identifications. The disclosedmethods of treating, monitoring, following-up with, advising, etc. canbe combined with any one or more other methods disclosed herein, and inparticular, with any one or more steps of the disclosed methods ofidentification.

EXAMPLES Example 1: Biomarkers on Melanoma Patient T Cells Associatedwith Ipilimumab Therapy

Materials and Methods

Patients

Between June 2004 and December 2008, 75 patients (39 at the Universityof Southern California, Norris Cancer Center and 36 at the MoffittCancer Center) with resected stage IIIC/IV melanoma received ipilimumabwith or without a peptide vaccine. The demographic and clinic outcomesof the patients for this study were shown in Table 1, which have beenreported previously (Sarnaik A A, et al. Clin Cancer Res. 17(4):896-906(2010)) with updated follow-up: three patients died (R DOD); one patientrelapsed and was resected with no evidence of disease (R NED) instead ofno evidence of disease (NED). Toxicity was assessed by National CancerInstitute Common Terminology Criteria for Adverse Events, version 3.0.The protocol was approved by the University of Southern California/LosAngeles County and University of South Florida Institutional ReviewBoards, and all patients provided written informed consent.

TABLE 1 Demographic data for the total 55 patients Variable Level n (%)dosage 10 mg/kg 40 (72.7)  3 mg/kg 15 (27.3) gender F 21 (38.2) M 34(61.8) stage III 24 (43.6) IV 31 (56.4) HLA_A2 A2+ 34 (61.8) A2− 21(38.2) irAE N 31 (56.4) Y 24 (43.6) Outcome NED 35 (63.6) Relapse 20(36.4)

PBMC Collection, Preparation and T Cells Purification

Apheresis with exchange of 5 to 7 liters was performed within 1 weekbefore and 6 months after the initiation of therapy, after four doses ofipilimumab. Heparinized blood samples were collected after 2 doses ofipilimumab, 3 months after the initiation of therapy. PBMC were isolatedfrom pre-, 3-month and 6-month post-ipilimumab treatment specimens byLymphoprep (Greiner Bio-One, Longwood, Fla.) density gradientcentrifugation. PBMC were counted, then frozen in 90% heat inactivatedhuman AB serum (HS, Omega Scientific, Tarzana, Calif.) and 10% dimethylsulfoxide (DMSO) (Sigma, St Louis, Mo.) and stored in secured liquidnitrogen freezers at −168° C. until use.

Cryopreserved pre- and 6-month post-treatment PBMC of each patient werethawed immediately into pre-warmed AIM-V media (Invitrogen Corporation,Grand Island, N.Y.) supplemented with 5% HS. For microarray analysis,PBMCs were sorted as follows: cells were stained with CD3-FITC (BDBioscience, San Diego, Calif.), CD4-PE (BD Bioscience), CD8-APC (BDBioscience) and CD19-PECy7 (BD Bioscience) for 30 min at 4° C. Afterextensive wash, PBMCs were stained with DAPI nucleic acid dye(Invitrogen) to exclude dead and dying cells. After washes with stainingbuffer (PBS+0.5% HS), PBMC were re-suspended in staining buffer for flowcytometry sorting. CD3⁺CD4⁺CD8⁻, CD3⁺CD8⁺CD4⁻ T cells and CD3⁻ CD19⁺ Bcell populations were collected by Aria (BD Bioscience, San Jose). Thepurity of the sorted specific cell populations were consistently ≥99%.

Microarray Analysis

The sorted cells were pelleted and re-suspended in a RLT lysis buffer(Qiagen Science, Valencia, Calif.), and delivered to the Microarray Core(Moffitt Cancer Center, Tampa, Fla.) for expression array analysis. RNAfrom the sorted cell populations were DNase-treated and extracted usingthe Qiagen RNeasy mini-prep according to the manufacturer's protocol(Qiagen). The Nugen Message Amp Premier kit was used to amplify 100nanograms of total RNA (Nugen Technologies, San Carlos, Calif.).Briefly, the extracted poly-(A) RNA was specifically converted to cDNA,amplified and labeled with biotin following the procedure initiallydescribed by Van Gelder et al. (Van Gelder R N, et al. Proc Natl AcadSci USA. 87:1663-7 (1990)). Biotin-labeled cDNA was hybridized ontoAffymetrix U133 Plus 2.0 microarrays. The hybridization, staining, andscanning of the chips followed the procedures outlined in the Affymetrixtechnical manual (Liu W M, et al. Bioinformatics. 18:1593-9 (2002)).Scanned output files were visually inspected for hybridization artifactsand then analyzed by using Affymetrix GeneChip Operating Software(GCOS). Arrays were scaled to an average intensity of 500 and analyzedindependently. The GCOS software used a statistical algorithm todetermine the signal intensity of a transcript from the behavior of 11different oligonucleotide probes designed to detect the same gene. Probesets that yielded a p-value less than 0.002 were identified as changed.Alternatively, signal intensity was calculated by the robust multi-arrayanalysis method (RMA) developed by Irizarry et al (Irizarry R A, et al.Nucleic Acids Res. 31:e15 (2003)). Gene changes were then selected usingthe fold-change cut-off available in RMA or using the SignificanceAnalysis of Microarrays (SAM) technique of Tusher et al. (Tusher V G, etal. Proc Natl Acad Sci USA. 98:5116-21 (2001)).

Surface and Intracellular Staining

For cell surface staining, 1-2×10⁶ PBMC in 100 μl PBS were stained withthe Aqua-live/dead fixable dye (Invitrogen) at 4° C. for 30 min. Afterwashing, PBMC were stained with fluorochrome-conjugated antibodiesagainst cell surface markers prepared as a master-mix-solution for 30min at 4° C. The antibodies for surface markers were purchased from BDBioscience unless otherwise specified: CD3-AlexFluor 700, CD4-PECy7,CD8-PERCPCy5.5, ICOS-PE, CCR7-FITC (R&D Systems, Minneapolis, Minn.),CCR7-PE (R &D Systems), CXCR3-FITC (R & D Systems), CD109-PE,CD45RA-FITC, CD28-APC (eBioscience, San Diego, Calif.), IL-7R-APC (R&Dsystem), CD25-FITC, TGFβR3-PE (R&D Systems) and MIC-A-APC (R&D Systems).The fluorescent minus one and isotype control was included in eachexperiment in order to appropriately set the gates. A total of 300,000live single lymphocytes were acquired on a LSR II flow cytometer (BDBioscience). Analysis gates were set on single live lymphocytes definedby scatter characteristics and Aqua negative staining. First, thelymphocytes were gated by the forward and sideward scatters, then thesingle cells were gated by forward and sideward width and height. Thesingle live lymphocytes were observed by gating on aqua negative stainedsingle lymphocytes. In the whole study, we observed the biomarkers onCD4 and CD8⁺ T cells.

For intracellular staining, 2-3×10⁶ PBMC in 100 μl staining buffer weretreated as above, then fixed with a freshly preparedFixation/Permeabilization working solution (eBioscience) at 4° C. for 30min. After wash with permeabilization buffer (eBioscience), the cellswere stained with intracellular markers: Ki67-FITC (Abcam, Cambridge,Mass.), EOMESodermin (EOMES)-Alex Fluor 647 (eBioscience), granzymeB-FITC, perforin-PE, FoxP3-APC (eBioscience), GATA 3-Alex Fluor 647(eBioscience), cleaved caspase III-PE for 30 min at 4° C. Cells werethen washed in the permeabilization buffer and resuspended in stainingbuffer for flow cytometry acquisition.

For the standardization of flow cytometry assays and the consistency ofdaily performance, a normal PBMC sample was stained and run on flowcytometry in parallel with the subject samples and Spherotech rainbowfluorescent particles (Spherotech, Tnc, Lake Forest, TL) were run beforerunning samples for alignment of the optical system of the flowcytometer in each channel.

Flow Cytometry Analysis

Flow cytometry data were analyzed using Flowjo software (Version 9.0.2,Tree Star, Inc., Asland, Oreg.). The mean fluorescence intensity (MFI)and frequency (%) of each marker was measured for CD4⁺ and CD8⁺ T cells.

Statistical Analysis

Descriptive summary statistics, that is, frequency and percentage fordiscrete variables and mean (standard deviation: STD), interquartilesand median (range) for continuous variables, were reported. Both pointestimates and their confidence intervals (CIs) were provided forparameters of interest, e.g., mean change or odds ratio (OR). Wilcoxonsigned-rank test was used to test whether changes in immunologicalbiomarker parameters between pre-, 3-month and 6-month post-ipilimumabwere statistically significant. Both absolute change and relativechange, i.e., post-pre and (post-pre)/pre*100, were analyzed to accountfor a potentially better normalization under the log scale for theimmunological biomarker parameters under investigation. Confidenceintervals were set at the 95% level. Univariable logistic regressionmodels were employed to explore the effect of absolute change as well asrelative change of each biomarker on disease outcome (NED vs. relapsed)and dose limiting irAE (Yes vs. No). The CI for OR based on theunivariable logistic regression model results were calculated for anincrement that equals one half of the interquartile range of thecorresponding change variable for the biomarker. A p-value of ≤0.05 wasconsidered statistically significant.

Results

Changes in Gene Expression Patterns of CD4⁺ and CD8⁺ T Cells Induced byIpilimumab

To assess the effect of ipilimumab on global gene expression in CD4⁺ andCD8⁺ T cells in vivo, microarray analysis was conducted on T cellspurified by flow cytometry from PBMCs collected from 12 resectedmelanoma patients pre- and 6-month post-ipilimumab treatment (Table 6a).The genes whose expression were most significantly altered in CD4⁺ andCD8⁺ T cells by ipilimumab treatment are listed in Tables 2a and 2b,categorized by T cell biology and function. The genes were grouped intocell cycle, cytokine-related, chemokine-related, T cell activation andproliferation signal-related, survival and apoptotic signal-related,kinase-related and heat shock protein family-related genes. It is worthyto note that the genes most impacted by ipilimumab were cell cyclerelated in both CD4⁺ and CD8⁺ T cells. Ipilimumab appeared to releasethe arrested cell cycle in CD4⁺ and CD8⁺ T cells as evidenced bysignificantly up-regulated CDC2 (Tables 2a and 2b), and other cell cyclerelated genes. Fold change was filtered as an increase ≥1.2 or adecrease ≤0.8-fold, and was statistically significantly different withp≤0.03 by paired Student T-test. The reported genes were allconsistently changed in ≥66.67% patients' pre and post ipilimumab byAffymetrix present/absent call.

TABLE 6a Patient Demographics for the Microarray cohort (n = 12)Variable Level n (%) gender F 4 (33.3) M 8 (66.7) stage III 4 (33.3) IV8 (66.7) HLA A2 A2+ 7 (58.3) A2− 5 (41.7) dosage 10 mg/kg 12 (100)  3mg/kg 0 (0) irAE N 7 (58.3) Y 5 (41.7) Outcome NED 12 (100) Relapse 0(0)

TABLE 2a genes impacted by ipilimumab in CD4+ T cells Gene symbol FoldGene name change p-value FDR Pre-Mean ± SD Post-Mean ± SD Cellcycle-related CDC2 1.98, 0.0090, 0.0409, 18.61 ± 4.41, 36.92 ± 21.36,cell division cycle 1.76, 0.0245, 0.0723, 7.05 ± 1.44, 12.41 ± 6.42, 2,G1 to S and G2 1.51 0.0189 0.0568 21.16 ± 6.79 36.37 ± 17.62 to M TYMS2.78, 0.0142, 0.0432, 66.68 ± 59.31, 185.59 ± 185.36, thymidylate 2.770.0075 0.0408 129.75 ± 58.10 358.99 ± 254.47 synthetase CCNB2 2.080.0142 0.0434 28.17 ± 11.75 58.49 ± 45.13 cyclin B2 CDK7 1.26 0.00740.0408 214.08 ± 75.01  270.13 ± 106.34 cyclin-dependent kinase 7Cytokines-related PGDS 2.2  0.0024 0.0393 14.20 ± 5.42  31.30 ± 14.57prostaglandin D2 synthase, hematopoietic TGFBR3 1.51, 0.0048, 0.04044,489.68 ± 470.87, 740.39 ± 581.83, transforming 1.39 0.0111 0.0411 505.71± 304.47 701.22 ± 246.45 growth factor b RIII IFNg 1.46 0.0255 0.0751138.35 ± 104.72 201.55 ± 120.78 interferon, g IL12A 1.33 0.001  0.037511.81 ± 2.00  15.73 ± 4.01  interleukin 12 A IL-7 1.28 0.0169 0.051115.43 ± 10.42 19.73 ± 8.89  interleukin 7 STAT1 1.28 0.0081 0.04091141.96 ± 330.11  1460.67 ± 577.64  signal transducer and activator oftranscription 1 SCYE1 1.27 0.0103 0.041  278.24 ± 100.74 352.82 ± 149.81small inducible cytokine subfamily E, member 1 IL-7R 0.80, 0.0061,0.0406, 5219.38 ± 1457.55, 4291.38 ± 1772.32, interleukin 7 0.74 0.00110.0375 3309.89 ± 1296.32 2467.81 ± 1094.22 receptor IL-2Ra 0.69  0.018110.0544 113.56 ± 58.89  79.12 ± 39.28 interleukin 2 receptor, a IL-150.74 0.0032 0.0398 54.76 ± 39.59 40.63 ± 31.09 interleukin 15 TNFSF80.73 0.0202 0.0605 248.08 ± 148.25 181.53 ± 92.03  tumor necrosis factorsuperfamily, member 8 Chemokines-related CCR3 1.52 0.0092 0.041  19.73 ±5.59  29.92 ± 13.46 Chemokine (C-C motif) receptor 3 ITGB1 1.29, 0.0055,0.0405, 1449.91 ± 772.02, 1867.82 ± 802.10, integrin, b1 1.20 0.00120.0375 396.68 ± 336.59 476.25 ± 376.51 (antigen CD29) CXCR3 1.29 0.02860.0825 47.04 ± 23.35 60.53 ± 33.18 chemokine receptor 3 CXCR7 0.750.0205 0.0614 83.62 ± 25.83 63.45 ± 25.78 chemokine receptor 7 ITGA60.72 0.0211 integrin, a6 Activation, proliferation anddifferentiation-related MKI67 1.98, 0.0165, 0.0498, 41.58 ± 22.80, 82.16± 62.67, antigen identified 1.69 0.0104 0.0410 19.37 ± 9.50 32.73 ±20.37 by monoclonal antibody Ki-67 ICOS 1.49 0.0011 0.0375 587.76 ±294.69 874.26 ± 397.84 inducible T-cell costimulator GATA3 1.43 0.01240.042  284.77 ± 189.82 406.03 ± 268.34 GATA binding protein 3 CTLA-41.37, 0.0023, 0.0392, 163.55 ± 83.74, 224.53 ± 114.73, cytotoxic T- 1.370.0249 0.0734 511.58 ± 260.28 700.23 ± 359.54 lymphocyte- associatedprotein 4 CD38 1.36 0.0075 0.0408 32.73 ± 13.37 44.62 ± 20.04 CD38molecule MICA 0.7  0.0015 0.0379 120.33 ± 67.61  84.36 ± 51.72 MHC classI polypeptide- related sequence A BCL family-related Bcl3 1.56 0.00360.0400 199.55 ± 112.65 311.36 ± 120.66 B-cell CLL/lymphoma 3 BAK1 1.262.00E−04 0.0365 130.50 ± 27.61  164.92 ± 33.45  Bcl2- antagonist/killer1 Bcl2L11 1.24, 0.0067, 0.0407, 314.36 ± 77.54, 388.54 ± 79.60,Bcl2-like 11 1.21 0.0369 0.1053 442.48 ± 299.18 536.88 ± 388.57 BCLAF11.21 0.0234 0.0695 61.14 ± 14.91 73.89 ± 26.64 Bcl2-associatedtranscription factor 1 Bcl2 0.79, 0.0246, 0.0726, 617.68 ± 266.67,492.80 ± 272.63, B-cell 0.72, 0.0158, 0.0479, 189.15 ± 81.00, 137.91 ±40.11, CLL/lymphoma 2 0.63 0.0050 0.0404 427.68 ± 327.43 272.97 ± 207.62Apoptosis MAP kinase and protein kinase-related ANXA5 1.32 0.0271 0.0794599.68 ± 269.08 793.89 ± 425.06 annexin 5 MAPK6 1.31 0.0261 0.0768209.49 ± 142.30 273.63 ± 202.36 mitogen-activated protein kinase 6PPP1CC 1.23 0.0152 0.0462 998.56 ± 432.59 1225.87 ± 571.01  proteinphosphatase 1, catalytic subunit, g isoform CASP7 1.22 0.0145 0.0441141.11 ± 41.72  171.83 ± 48.77  caspase 7, apoptosis-related cysteinepeptidase MAP2K6 0.77 0.0105 0.0410 77.3 ± 41.24 59.77 ± 33.17mitogen-activated protein kinase kinase kinase 6 ANXA11 0.75 0.00030.0368 93.23 ± 20.45 70.21 ± 31.98 annexin 11 ATF7 0.75 0.0252 0.0743170.44 ± 120.64 128.30 ± 107.21 activating transcription factor 7 MAP4K40.63 0.0095 0.041  137.49 ± 51.60  86.97 ± 29.96 mitogen-activatedprotein kinase kinase kinase 4 CASP10 0.62 0.004  0.0401 62.63 ± 27.1239.11 ± 15.88 caspase 10, apoptosis-related cysteine peptidase

TABLE 2b genes impacted by ipilimumab in CD8+ T cells Gene symbol Genename Fold change p-value FDR Pre-Mean ± SD Post-Mean ± SD Cellcycle-related CDCA2 1.95 0.0189 0.0647  12.86 ± 6.07  25.11 ± 16.20 celldivision cycle associated 2 CDC2 1.81, 0.0300, 0.0897,  18.37 ± 6.09, 33.23 ± 21.15, cell division cycle 2, 1.64 0.0274 0.0827  20.92 ± 6.16 34.31 ± 21.02 G1 to S and G2 to M CDCA7 1.67 0.0113 0.0647  87.63 ±93.37 146.50 ± 111.23 Cell division cycle associated 7 Cytokines-relatedTNFSF4 0.58 0.0082 0.0647  40.88 ± 27.10  23.93 ± 14.71 tumor necrosisfactor superfamily, member 4 Chemokines-related CXCR3 1.25 0.0036 0.0647 54.98 ± 21.50  68.96 ± 21.37 chemokine (C-X-C motif) receptor 3 ITGAV0.80 5.00E−04 0.0647  114.65 ± 54.10  91.78 ± 42.91 integrin, aV(antigen CD51) CXCR7 0.73 0.0268 0.081  92.65 ± 43.71  68.45 ± 33.28chemokine (C-X-C motif) receptor 7 Activation Differentiation andInteraction-related HLA-DRB4 1.4 0.0163 0.0647  28.04 ± 26.29  39.18 ±37.40 major histocompatibility complex, class II, DR b4 GATA3 1.39,0.0100, 0.0647,  178.84 ± 104.07, 248.93 ± 148.36, GATA binding protein1.35 0.0026 0.0647  139.84 ± 44.14 188.83 ± 75.51 3 HLA-DRA 1.38 0.01930.0647  540.53 ± 402.57 745.47 ± 350.32 major histocompatibilitycomplex, class II, DR a CD6 1.32 0.0248 0.0754  179.47 ± 95.79 236.58 ±157.76 CD6 molecule (CD166 receptor) CD5 1.22 0.0181 0.0647  103.58 ±38.21 126.47 ± 45.17 CD5 molecule CD209 1.20 0.0288 0.0865  36.73 ± 4.88 44.08 ± 7.60 CD209 molecule EOMES 0.69 0.0292 0.0877 1063.26 ± 642.42738.05 ± 445.38 Eomesodermin Apoptosis, MAP Kinases-related TIAM1 1.680.0105 0.0647  83.63 ± 67.00 140.61 ± 116.68 T-cell lymphoma invasionand metastasis 1 ANXA5 1.31 0.0114 0.0647  576.93 ± 278.33 747.53 ±316.78 annexin 5 API5 1.24 0.0088 0.0647  187.92 ± 118.48 233.04 ±151.93 apoptosis inhibitor 5

Pharmacodynamic Effects of Ipilimumab on T Cells

To verify changes in selected molecules from the microarray analysis andfurther investigate the mechanism of action of ipilimumab, a flowcytometry study was undertaken with the pre-, 3-month and 6-monthpost-ipilimumab PBMCs from expanded groups of 25 and 37 patientsrespectively (Table 6b, 6c) overlapping the microarray cohort of 12. Inaddition to the selected immunological biomarkers, CD4⁺ and CD8⁺ T celleffector-memory/naive phenotypes were also measured. Biomarkers with MFIand frequency both significantly changed in absolute value and foldchange with p≤0.02 by Wilcoxon and also significant by paired StudentT-test.

TABLE 6b Demographics of patients with 3-month measurements (n = 25)Variable Level n (%) gender F 9 (36.0) M 16 (64.0) stage III 10 (40.0)IV 15 (60.0) HLA A2 A2+ 18 (72.0) A2− 7 (28.0) dosage 10 mg/kg 16 (64.0) 3 mg/kg 9 (36.0) irAE N 15 (60.0) Y 10 (40.0) Outcome NED 17 (68.0)Relapse 8 (32.0)

TABLE 6c Demographics of patients with 6-month measurements (n = 37)Variable Level n (%) gender F 12 (32.4) M 25 (67.6) stage III 17 (45.9)IV 20 (54.1) HLA A2 A2+ 20 (54.1) A2− 17 (45.9) dosage 10 mg/kg 31(83.8)  3 mg/kg  6 (16.2) irAE N 23 (62.2) Y 14 (37.8) Outcome NED 27(73.0) Relapse 10 (27.0)

Increased Ki67 Expression in, and ICOS on CD4^(|) and CD8^(|) T Cells 3-and 6-Months after Ipilimumab

The Ki-67 protein (also known as MKI67) is a cellular marker forproliferation (Scholzen T, et al. J Cell Physiol. 182:311-22 (2000)) andcell cycling (Hertoghs K M, et al. J Clin Invest. 120:4077-90 (2010))and is an indicator of the growth fraction of a given cell population.As demonstrated in Table 3a and Table 3b, the MFI and frequency of Ki67in CD4⁺ and CD8⁺ T cells was significantly increased in both 3-month(Table 3a) and 6-month (Table 3b) post-treatment PBMCs with p≤0.0032 forall by Wilcoxon.

TABLE 3a Statistical analysis of changes in biomarkers at 3 monthsMedian Median fold change* Wilcoxon change* Wilcoxon Biomarker n (Q1,Q3) p (Q1, Q3) p MFI-ICOS-CD4 25    2.10.00 <.0001 1.41 <.0001  (71.00,273.00) (0.60, 2.14) Frequency-ICOS- 25 13.00 <.0001 1.35 <.0001 CD4 (1.15, 20.69) (0.22, 3.62) MFI-ICOS-CD8 25 34.90 <.0001 0.56 <.0001 (4.60, 46.10) (0.09, 1.12) Frequency-ICOS- 25  4.19 <.0001 2.90 <.0001CD8 (2.07, 7.75) (1.25, 6.71) MFI-Ki67-CD4 24 779.50  <.0001 0.58 <.0001 (178.50, 1657.00) (0.12, 1.43) Frequency-Ki67- 24  3.62 <.0001 1.49<.0001 CD4 (0.74, 6.94) (0.18, 2.63) MFI-Ki67-CD8 24 480.00  0.0032 0.320.0011   (−6.00, −1268.00) (−0.00, 1.23)  Frequency-Ki67- 24  1.490.0009 0.54 0.0003 CD8 (−0.10, 4.86)  (−0.03, 2.20)  MFI-CCR7-CD8 21−279.00  0.0003 −0.23  0.0004 (−645.00, 131.00)  (−0.30, 0.12) Frequency-CCR7- 25 −3.98 0.0122 −0.14  0.018 CD8 (−11.40, 1.30)  (−0.27,0.04) 

TABLE 3b Statistical analysis of changes in biomarkers at 6 monthsMedian Median fold change* Wilcoxon change* Wilcoxon Biomarker n (Q1,Q3) p (Q1, Q3) p MFI-ICOS-CD4 37 111.00  <.0001 0.84 <.0001  (46.80,166.00) (0.41, 1.06) Frequency-ICOS- 37 9.45 <.0001 1.66 <.0001 CD4 (3.13, 14.57) (0.70, 2.42) MFI-ICOS-CD8 37 29.10  <.0001 0.47 <.0001(11.80, 44.50) (0.21, 0.64) Frequency-ICOS- 37 3.07 <.0001 1.34 <.0001CD8 (1.62, 5.36) (0.63, 3.30) MFI-Ki67-CD4 36 535.50  <.0001 0.49 <.0001 (207.50, 1517.50) (0.24, 1.16) Frequency-Ki67- 35 2.33 <.0001 0.87<.0001 CD4 (1.37, 6.97) (0.29, 2.12) MFI-Ki67-CD8 36 326.00  <.0001 0.30<.0001  (85.50, 770.50) (0.10, 0.70) Frequency-Ki67- 36 1.88 0.0004 0.55<.0001 CD8 (0.13, 4.91) (0.04, 1.52) MFI-Gata3-CD4 28 78.00  0.004 0.210.0038  (8.00, 189.50) (−0.02, 0.43)  Frequency-Gata3- 28 3.76 0.00040.94 <.0001 CD4 (0.28, 8.04) (−0.01, 1.62)  MFI-Gata3-CD8 28 76.50 0.0045 0.16 0.0032  (8.00, 119.50) (0.03, 0.35) Frequency-Gata3- 28 2.070.0006 0.59 <.0001 CD8 (0.59, 4.02) (0.09, 1.23) MFI-CCR7-CD4 37−290.00   <.0001 −0.14  <.0001 (−587.00, −113.00) (−0.21, −0.07)Frequency-CCR7- 37 −2.90  0.0017 −0.03  0.006 CD4 (−6.80, −0.40) (−0.09,0.01)  MFI-IL-7R-CD4 37 −23.00  0.0001 −0.08  <.0001 (−45.00, −7.00) (−0.17, 0.02)  Frequency-IL-7R- 37 −1.70  0.0093 −0.15  0.0271 CD4(−3.20, 0.36)  (−0.34, 0.05) 

Inducible T-cell co-stimulator (ICOS) is a T cell surface moleculestructurally related to CD28 and CTLA-4 (Dong C, et al. Nature.409:97-101 (2001)). It is expressed at low levels on resting naïve Tcells and is rapidly up-regulated following TCR ligation and CD28costimulation (McAdam A J, et al. J Immunol 165:5035-40 (2000)). Afteripilimumab treatment, the MFI and frequency of ICOS was increasedsignificantly on CD4⁺ and CD8⁺ T cells, with higher increases on CD4⁺than on CD8⁺ T cells and higher increases at 3-month than at 6-monthpost treatment (Table 3).

Decreased CCR7 on CD8^(|) and IL-7R Expression on CD4⁺ Tcells afterIpilimumab

The MFI and frequency of CCR7 expression on CD8⁺ T cells was decreasedat 3- and 6-month post-ipilimumab (Table 3). The MFI of IL-7R on CD4⁺ Tcells was down-regulated at 3-month and 6-month post-ipilimumabtreatment, and the frequency of IL-7R on CD4+ T cells was significantlydecreased at 6-month post-ipilimumab treatment (Table 3).

Increased GATA3 Expression in CD4⁺ and CD8⁺ T Cells

GATA3 is a transcription factor that is a marker for Th2 polarizationand is associated with the generation of Th2 cytokines IL-4, IL-5 andIL-10. After treatment with ipilimumab, GATA3 expression as MFI andfrequency was increased significantly in CD4^(|) and CD8^(|) T cells at6-month post-ipilimumab treatment (Table 3b).

Surrogate Biomarkers on/in T Cells Associated with Relapse or irAE

In an univariate logistic regression analysis using changes inexpression by flow cytometry as a continuous variable one at a time,only absolute decrease in Ki67⁺EOMES⁺CD4⁺, in MFI and frequency ofCCR7⁺CD8⁺, and increase in MFI of TGFβR3 on CD8⁺, in MFI and frequencyof EOMES⁺CD8⁺ and in GranzymeB⁺EOMES⁺CD8⁺ T cells, Perforin⁺EOMES⁺CD8⁺ Tcells, and CTLA-4⁺CD4⁺ T cells were associated with a higher likelihoodof relapse with p<0.05 (Table 4a). Only absolute decrease of Ki67⁺CD8⁺ Tcells, CD71⁺CD8⁺ T cells, ICOS⁺CD8⁺ T cells, and PD-1⁺CD8⁺ T cells wereassociated with a development of irAE, (Table 4b) with p<0.05.

TABLE 4a Association of changes in biomarkers at 6 months with outcomeVariable Slope p-value Ki67 EOMES CD4 (%) −3.5232 0.0285 MFI CCR7 CD8−0.0021 0.0158 CCR7 CD8 (%) −0.0947 0.0379 EOMES CD8 (%) 0.1758 0.0072MFI EOMES CD8 0.0025 0.0282 MFI TGFbRS CD8 0.0049 0.0337 EOMES PerforinCD8 (%) 0.2109 0.0278 EOMES Granzyme B CD8 (%) 0.2126 0.0078 CTLA-4 CD4(%) 0.4222 0.0178

TABLE 4b Association of changes in biomarkers with irAE (Yes vs. No)Variable Slope p-value Ki67 CD8 (%) −0.3167 0.0217 CD71 CD8 −0.54750.0484 CD71 CD8 (%) −0.0075 0.0297 ICOS CD8 (%) −0.0049 0.0411 PD-1 CD8(%) −0.0293 0.0476 (%) means percentage of CD4⁺ or CD8⁺ T cells (asindicated) that had the indicated markers. MFI means the meanfluorescence intensity of the indicated marker in the indicated type ofT cell (CD4⁺ or CD8^(+).)

Pre-Treatment Biomarkers on/in T Cells Associated with Outcome and irAE

An analysis dichotomized by the median of baseline biomarkers revealedthat low Ki67⁺EOMES⁺CD8⁺, high MFI of CCR7⁺CD4⁺ and low EOMES⁺CD8⁺ weresignificantly associated with relapse (Table 5a) with p values of0.0008, 0.0190 and 0.0242 respectively. These pre-treatment biomarkerscollected from a cohort of 55 patients were also confirmed in aunivariable logistic regression analysis. Another analysis of covariatedichotomized by the median value for baseline biomarkers showed that lowKi67⁺EOMES⁺CD4⁺ and high MFI of TGFβR3 on CD8⁺ T cells were associatedwith occurrence of irAE (Table 5b) with p=0.0046 and p=0.0218respectively.

TABLE 5a Association between outcome and dichotomized baselinebiomarkers Chi- Out- Covariate Odds Ratio Square Association come Leveln (%) (95% CI) p-value Ki67EOMES CD8 Relapse ≤2.11 15 (75.0) 11.250.0008 Outcome NED ≤2.11 5 (25.0)  (2.52, 50.27) Relapse >2.11 4 (21.1)NED >2.11 15 (78.9) MFI CCR7 CD4 Relapse ≤2402 6 (21.4) 0.25 0.019Outcome NED ≤2402 22 (78.6) (0.08, 0.82) Relapse >2402 14 (51.9)NED >2402 13 (48.1) Frequency EOMES Relapse ≤55.6 14 (51.9) 3.77 0.0242CD8 Outcome NED ≤55.6 13 (48.1)  (1.16, 12.27) Relapse >55.6 6 (22.2)NED >55.6 21 (77.8) Frequency CD109 Relapse ≤0.7315 5 (19.2) 0.20 0.0201CD8 Outcome NED ≤0.7315 21 (80.8) (0.06, 0.71) Relapse >0.7315 14 (53.8)NED >0.7315 12 (46.2)

TABLE 5b Association between irAE (Yes/No) and baseline dichotomizedbiomarkers Chi- Out- Covariate Odds Ratio Square Association come Leveln (%) (95% CI) p-value Ki67EOMES Y ≤0.446 12 (60.0) 8.00 0.0046 CD4 irAEN ≤0.446 8 (40.0) (1.74, 36.70) Y >0.446 3 (15.8) N >0.446 16 (84.2) MFITGFbR3 Y ≤527 8 (28.6) 0.28 0.0218 CD8 irAE N ≤527 20 (71.4) (0.09,0.85)  Y >527 16 (59.3) N >527 11 (40.7) Frequency Y ≤2.79 18 (69.2)7.50 0.0019 CD71 N ≤2.79 8 (30.8) (2.18, 25.80) CD4 irAE Y >2.79 6(23.1) N >2.79 20 (76.9)

This analysis pointed out the potential importance of EOMES, atranscription factor in the T-box family and involved in the regulationof INF-γ, granzyme B and perforin production by CD8⁺ T cells (Pearce EL, et al. Science. 302:1041-3 (2003)). To better understand thepotential role of EOMES⁺CD8⁺ T cells in ipilimumab treatment,pre-treatment specimens were stratified by the median frequency ofEOMES⁺ in CD8⁺ T cells. High baseline frequency of EOMES⁺ in CD8⁺ Tcells were significantly associated with an improved relapse freesurvival (RFS) compared with a lower basal level of EOMES⁺CD8⁺ T cells(P=0.0183; log-rank test) (FIG. 1A). The patients were stratified by themedian frequency of Ki67⁺EOMES⁺CD8⁺. The patients with a higherproportion of Ki67⁺EOMES⁺CD8⁺ T cells had significantly improved RFScompared with those patients with lower frequency of Ki67⁺EOMES⁺CD8⁺cells (FIG. 1B), p=0.0004 by the long-rank test. Patients were alsostratified by the median MFI of CCR7 on CD4⁺ T cells. The patients withlower MFI of CCR7⁺ on CD4⁺ had a significantly better RFS than thehigher expressing patients (FIG. 1C), with p value of 0.0278 by thelog-rank test.

Some of the significant biomarkers were tested on the 11 availablenormal phereses. Results for the most significant biomarker(EOMES⁺Ki67⁺CD8⁺) on the 11 normals and the melanoma patients (relapseand NED) in this trial is shown in FIG. 2. The median of EOMES⁺Ki67⁺CD8⁺T cells for the 11 normal was 1.57, very similar to the median for therelapse group patients which was 1.50. For the NED group, the median ofEOMES⁺Ki67⁺CD8⁺ T cells was 2.49 significantly higher than median ofrelapsed patients (p=0.03, student T test).

Biomarker levels associated with cancer prognosis and CTLA-4 blockadetreatment are shown in Table 7. The range in the last column is therange of values for that biomarker seen in the group of patients.

TABLE 7 Biomarker Levels Associated with Cancer Prognosis and CTLA-4Blockade Treatment Range for Cut off total Biomarkers Association UnitValue patients Ki67+EOMES+CD8+ outcome-NED % >2.11 5.77 EOMES+CD8+outcome-NED % >55.6 67.8 MFI-CCR7-CD4+ outcome-NED MFI ≤2402 3080Ki67+EOMES+CD4+ irAE-No % >0.446 1.96 MFI-TGFbR3-CD8+ irAE-No MFI ≤5271711 * %: Percentage * MFI: Mean Fluorescence Intensity

Example 2: Gene Profiling of Melanoma Patients CD8+ T Cells Associatedwith PD-1 Blockade Treatment and Clinical Outcome

PD-1 blocking antibody (BMS-936558) therapy has shown antitumor activityand clinical benefit in melanoma patients. The precise molecular basisand mechanisms of PD-1 blockade in vivo have not been documented andthere are few biomarker associated with clinical benefit. The purpose ofthis study is to understand the mechanisms and investigate potentialbaseline and surrogate biomarkers associated with clinical benefit ofPD-1 blockade in stage IV unresectable melanoma patients' T cells invivo.

Materials and Methods

Microarray analysis was performed on flow sorted CD8⁺ T cells from PBMCscollected pre- and 12 week-post PD-1 blockade treatment at 1, 3 and 10mg/kg. The pre-treatment sample was collected within 1 week before antiPD-1 treatment. The post-treatment sample was collected 12 weeks afterthe initial treatment with 6 injections of anti PD-1 antibody. StageIIIC and stage IV melanoma patients received intravenous infusion ofanti PD-1 (BMS-936558) in dose-escalating ten-patient cohorts at 1, 3,10 mg/kg.

Results

To address the molecular mechanisms of PD-1 blockade on human CD8⁺ Tcells, the molecular signature of PD-1 blockade treatment wascharacterized at 1, 3, 10 mg/kg by comparing the gene-expressionprofiles of CD8⁺ T cells from pre and post treatment T cells.

Gene signatures associated with clinical benefit were assessed bycomparing 11 clinical responders to 11 non-responders at baseline,post-treatment at 12 weeks and assessed changes of post vs. pre samples.

Results: PD-1 blockade differentially impacts gene expression of CD8⁺ Tcells at 1, 3 and 10 mg/kg. Baseline and post treatment gene profiles ofCD8⁺ T cells are significantly associated with clinical benefit (FIG. 4(PCA), FIGS. 5 and 6 (Heat Map)). At base line: low CD276 (B7-H3), highNKTR, PTEN and GATAD1 in CD8⁺ T cells are associated with clinicalresponse (Table 9). Increased LAG1, decreased GADD45B, EGR1 and EGR2 areassociated with clinical benefit at week 12 post-treatment (Table 8).

Baseline, post-treatment, and changes in gene expression profiling ofCD8⁺ T cells are significantly associated with clinical response afterPD-1 blockade. This approach revealed key insights into the biology ofPD-1 blockade. Breaking CD8⁺ T cell anergy is thus associated withclinical benefit.

TABLE 8 Post-treatment Changes in Gene Profile Associated with ResponseMean Post/ Symbol Gene Title Fold pValue Mean Pre LASS6 LAG1 homolog,1.30 0.0211 263.9/202.6 ceramide synthase 6 GADD45G growth arrest, 0.790.0414  90.1/114.0 DNA-damage- inducible, g ING1 inhibitor of growth0.78 0.016 100.4/128.0 family, member 1 CKS2 CDC28 protein kinase 0.780.0101 345.3/445.0 regulatory subunit 2 CDKN1C Cyclin-dependent kinase0.78 0.0010 65.8/84.5 inhibitor 1C IFIH1 interferon induced with 0.780.0305 38.3/48.9 helicase C domain 1 GADD45B Growth arrest 0.67 0.00031851.1/2647.7 DNA-damage- inducible, beta IER5 immediate early 0.760.0045 3906.4/5155.9 response 5 IFNG interferon, gamma 0.73 0.02321062.9/1446.8 IER5L immediate early 0.67 0.0207 180.9/271.3 response5-like EGR2 early growth response 2 0.67 0.046  81.9/121.9 EGR1 Earlygrowth response 1 0.35 0.0156 1119.0/3218.2

TABLE 9 Baseline Gene Expression Associated with Response MeanResponders/ Symbol Gene Title Fold pValue Non Responders USP9Y ubiquitinspecific peptidase 9, 2.23 0.0441 255.2/114.3 Y-linked HSPD1 heat shock60 kDa protein 1 1.92 0.0415 50.8/26.4 (chaperonin) NSUN6 NOL1/NOP2/Sundomain 1.72 3.04E−05 150.5/87.3  family, member 6 CISH cytokineinducible SH2- 1.7 0.0288 248.1/145.5 containing protein KLF12Kruppel-like factor 12 1.59 0.0142 192.5/121.3 NCF4 neutrophil cytosolicfactor 4, 1.51 0.049 59.8/39.5 40 kDa SFRS7 splicing factor, 1.49 0.01131000.1/670.6  arginine/serine-rich 7, 35 kDa CDC14A CDC14 cell divisioncycle 14 1.49 0.0412 144.3/97   homolog A (S. cerevisiae) MAP2K5Mitogen-activated protein 1.48 0.017 77.4/52.4 kinase kinase 5 CCAR1Cell division cycle and 1.46 0.0095 241.3/165.2 apoptosis regulator 1IL11RA interleukin 11 receptor, alpha 1.46 0.0232 604.3/414.7 MLLmyeloid/lymphoid or mixed- 1.43 0.0391 36.2/25.3 lineage leukemiaHSPBAP1 HSPB (heat shock 27 kDa) 1.42 0.0113 319.2/225.2 associatedprotein 1 MAP4K4 mitogen-activated protein 1.41 0.0242 223.2/158.7kinase kinase kinase kinase 4 HELLS helicase, lymphoid-specific 1.410.0351 107.4/76.0  SMYD2 SET and MYND domain 1.37 0.0009 168.2/122.6containing 2 C1QTNF3 C1q and tumor necrosis 1.36 0.0242 65.6/48.1 factorrelated protein 3 PTEN phosphatase and tensin 1.34 0.0232 49.9/37.3homolog UBE2D1 ubiquitin-conjugating enzyme 1.34 0.0418 77.4/57.8 E2D 1FBXO9 F-box protein 9 1.33 0.006 208.1/156.7 FAIM3 Fas apoptoticinhibitory 1.33 0.0164 885.2/665.9 molecule 3 IFI44 Interferon-inducedprotein 44 1.33 0.043 91.8/68.9 NCRNA00153 Non-protein coding RNA 1531.31 0.0031 146.3/111.8 NSMAF Neutral sphingomyelinase 1.3 0.0014149.4/114.8 activation associated factor H2BFM H2B histone family,member 1.3 0.0412 100.5/77.5  M GATAD1 GATA zinc finger domain 1.290.0412 214.7/166.5 containing 1 GATAD1 GATA zinc finger domain 1.270.0258 385.9/302.9 containing 1 NKTR natural killer-tumor 1.27 0.02632486.9/1951.9 recognition sequence CASP8 caspase 8, apoptosis-related1.27 0.035 2326.9/1839.1 cysteine peptidase SOCS1 suppressor of cytokine0.8 0.0182  95.7/119.9 signaling 1 P2RY4 pyrimidinergic receptor P2Y,0.8 0.0005 39.4/49.1 G-protein coupled, 4 CD276 CD276 molecule (B7-H3)0.79 0.0003 108.1/136.9 RCOR2 REST corepressor 2 0.78 0.0011 156.9/201.4PDCD6 Programmed cell death 6 0.77 0.0151  921.4/1198.5 NUB1 negativeregulator of 0.75 0.0054  84.1/111.4 ubiquitin-like proteins 1 FCRL2 Fcreceptor-like 2 0.71 0.0045 62.1/87.8 NUCKS1 Nuclear casein kinase,cyclin- 0.69 0.0055 24.5/35.4 dependent kinase substrate 1 SHC2 SHCtransforming protein 2 0.66 0.0036  98.1/149.1 SCD5 stearoyl-CoAdesaturase 5 0.64 0.0037 211.6/328.3

Disclosed are materials, compositions, and components that can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed method and compositions. These and othermaterials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials aredisclosed that while specific reference of each various individual andcollective combinations and permutation of these compounds may not beexplicitly disclosed, each is specifically contemplated and describedherein. For example, if a PD-1 blockade therapy is disclosed anddiscussed and a number of modifications that can be made to a number ofmolecules including the active molecule are discussed, each and everycombination and permutation of the therapy and molecule and themodifications that are possible are specifically contemplated unlessspecifically indicated to the contrary. Thus, if a class of molecules A,B, and C are disclosed as well as a class of molecules D, E, and F andan example of a combination molecule, A-D is disclosed, then even ifeach is not individually recited, each is individually and collectivelycontemplated. Thus, is this example, each of the combinations A-E, A-F,B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated andshould be considered disclosed from disclosure of A, B, and C; D, E, andF; and the example combination A-D. Likewise, any subset or combinationof these is also specifically contemplated and disclosed. Thus, forexample, the subgroup of A-E, B-F, and C-E are specifically contemplatedand should be considered disclosed from disclosure of A, B, and C; D, E,and F; and the example combination A-D. Further, each of the materials,compositions, components, etc. contemplated and disclosed as above canalso be specifically and independently included or excluded from anygroup, subgroup, list, set, etc. of such materials. These concepts applyto all aspects of this application including, but not limited to, stepsin methods of making and using the disclosed compositions. Thus, ifthere are a variety of additional steps that can be performed it isunderstood that each of these additional steps can be performed with anyspecific embodiment or combination of embodiments of the disclosedmethods, and that each such combination is specifically contemplated andshould be considered disclosed.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, also specifically contemplated and considered disclosed isthe range from the one particular value and/or to the other particularvalue unless the context specifically indicates otherwise. Similarly,when values are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value forms another,specifically contemplated embodiment that should be considered disclosedunless the context specifically indicates otherwise. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint unless the context specifically indicates otherwise. Finally,it should be understood that all of the individual values and sub-rangesof values contained within an explicitly disclosed range are alsospecifically contemplated and should be considered disclosed unless thecontext specifically indicates otherwise. The foregoing appliesregardless of whether in particular cases some or all of theseembodiments are explicitly disclosed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed method and compositions belong. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present method andcompositions, the particularly useful methods, devices, and materialsare as described. Publications cited herein and the material for whichthey are cited are hereby specifically incorporated by reference.Nothing herein is to be construed as an admission that the presentinvention is not entitled to antedate such disclosure by virtue of priorinvention. No admission is made that any reference constitutes priorart. The discussion of references states what their authors assert, andapplicants reserve the right to challenge the accuracy and pertinency ofthe cited documents. It will be clearly understood that, although anumber of publications are referred to herein, such reference does notconstitute an admission that any of these documents forms part of thecommon general knowledge in the art.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. A method comprising assaying peripheral blood mononuclearcells (PBMCs) from a subject diagnosed with melanoma for expression ofCD8 and one or a combination of Phosphatase and tensin homolog (PTEN),GATA zinc finger domain containing 1 (GATAD1), Natural killer-tumorrecognition sequence (NKTR), CD276 molecule (B7-H3) (CD276),NOL1/NOP2/Sun domain family, member 6 (NSUN6), Cell division cycle andapoptosis regulator 1 (CCAR1), SET and MYND domain containing 2 (SMYD2),F-box protein 9 (FBXO9), Non-protein coding RNA 153(NCRNA00153), Neutralsphingomyelinase activation associated factor (NSMAF), Pyrimidinergicreceptor P2Y, G-protein coupled, 4 (P2RY4), REST corepressor 2 (RCOR2),Negative regulator of ubiquitin-like proteins 1 (NUB1), Fc receptor-like2 (FCRL2), Nuclear casein kinase, cyclin-dependent kinase substrate 1(NUCKS1), SHC transforming protein 2 (SHC2), Stearoyl-CoA desaturase 5(SCD5), Ubiquitin specific peptidase 9, Y-linked (USP9Y), Heat shock 60kDa protein 1 (chaperonin) (HSPD1), Cytokine inducible SH2-containingprotein (CISH), Kruppel-like factor 12 (KLF12), Neutrophil cytosolicfactor 4, 40 kDa (NCF4), Splicing factor, arginine/serine-rich 7, 35 kDa(SFRS7), CDC14 cell division cycle 14 homolog A (S. cerevisiae)(CDC14A), Mitogen-activated protein kinase kinase 5 (MAP2K5),Interleukin 11 receptor, alpha (IL11RA), Myeloid/lymphoid ormixed-lineage leukemia (MLL), HSPB (heat shock 27 kDa) associatedprotein 1 (HSPBAP1), Mitogen-activated protein kinase kinase kinasekinase 4 (MAP4K4), Helicase, lymphoid-specific (HELLS), C1q and tumornecrosis factor related protein 3 (C1QTNF3), Ubiquitin-conjugatingenzyme E2D 1 (UBE2D1), Fas apoptotic inhibitory molecule 3 (FAIM3),Interferon-induced protein 44 (IFI44), H2B histone family, member M(H2BFM), GATA zinc finger domain containing 1 (GATAD1), Caspase 8,apoptosis-related cysteine peptidase (CASP8), Suppressor of cytokinesignaling 1 (SOCS1), Programmed cell death 6 (PDCD6), LAG homolog,ceramide synthase 6 (LASS6), Growth arrest DNA-damage-inducible, beta(GADD45B), Early growth response 2 (EGR2), Early growth response 1(EGR1), Growth arrest, DNA-damage-inducible, g (GADD45G), CDC28 proteinkinase regulatory subunit 2 (CKS2), Cyclin-dependent kinase inhibitor 1C(CDKN1C), Immediate early response 5 (IER5), Inhibitor of growth family,member 1 (ING1), Interferon induced with helicase C domain 1 (IFIH1),Interferon, gamma (IFNG), Immediate early response 5-like (IER5L),Cancer/testis antigen 1 (NY-ESO-1), and Melanoma antigen recognized byT-cells 1 (MART-1); wherein the expression level of PTEN on CD8⁺ T cellsin the PBMCs is positively associated with the expected clinical benefitof PD-1 blockade treatment of the subject, wherein the expression levelof GATAD1 on CD8⁺ T cells in the PBMCs is positively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject,wherein the expression level of NKTR on CD8^(|) T cells in the PBMCs ispositively associated with the expected clinical benefit of PD-1blockade treatment of the subject, wherein the expression level of CD276on CD8⁺ T cells in the PBMCs is negatively associated with the expectedclinical benefit of PD-1 blockade treatment of the subject, wherein theexpression level of NSUN6 on CD8⁺ T cells in the PBMCs is positivelyassociated with the expected clinical benefit of PD-1 blockade treatmentof the subject, wherein the expression level of CCAR1 on CD8⁺ T cells inthe PBMCs is positively associated with the expected clinical benefit ofPD-1 blockade treatment of the subject, wherein the expression level ofSMYD2 on CD8⁺ T cells in the PBMCs is positively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject,wherein the expression level of FBXO9 on CD8⁺ T cells in the PBMCs ispositively associated with the expected clinical benefit of PD-1blockade treatment of the subject, wherein the expression level ofNCRNA00153 on CD8⁺ T cells in the PBMCs is positively associated withthe expected clinical benefit of PD-1 blockade treatment of the subject,wherein the expression level of NSMAF on CD8⁺ T cells in the PBMCs ispositively associated with the expected clinical benefit of PD-1blockade treatment of the subject, wherein the expression level of P2RY4on CD8⁺ T cells in the PBMCs is negatively associated with the expectedclinical benefit of PD-1 blockade treatment of the subject, wherein theexpression level of RCOR2 on CD8⁺ T cells in the PBMCs is negativelyassociated with the expected clinical benefit of PD-1 blockade treatmentof the subject, wherein the expression level of NUB1 on CD8⁺ T cells inthe PBMCs is negatively associated with the expected clinical benefit ofPD-1 blockade treatment of the subject, wherein the expression level ofFCRL2 on CD8^(|) T cells in the PBMCs is negatively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject,wherein the expression level of NUCKS1 on CD8⁺ T cells in the PBMCs isnegatively associated with the expected clinical benefit of PD-1blockade treatment of the subject, wherein the expression level of SHC2on CD8⁺ T cells in the PBMCs is negatively associated with the expectedclinical benefit of PD-1 blockade treatment of the subject, wherein theexpression level of SCD5 on CD8⁺ T cells in the PBMCs is negativelyassociated with the expected clinical benefit of PD-1 blockade treatmentof the subject, wherein the expression level of USP9Y on CD8⁺ T cells inthe PBMCs is positively associated with the expected clinical benefit ofPD-1 blockade treatment of the subject, wherein the expression level ofHSPD1 on CD8⁺ T cells in the PBMCs is positively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject,wherein the expression level of CISH on CD8⁺ T cells in the PBMCs ispositively associated with the expected clinical benefit of PD-1blockade treatment of the subject, wherein the expression level of KLF12on CD8⁺ T cells in the PBMCs is positively associated with the expectedclinical benefit of PD-1 blockade treatment of the subject, wherein theexpression level of NCF4 on CD8⁺ T cells in the PBMCs is positivelyassociated with the expected clinical benefit of PD-1 blockade treatmentof the subject, wherein the expression level of SFRS7 on CD8⁺ T cells inthe PBMCs is positively associated with the expected clinical benefit ofPD-1 blockade treatment of the subject, wherein the expression level ofCDC14A on CD8⁺ T cells in the PBMCs is positively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject,wherein the expression level of MAP2K5 on CD8^(|) T cells in the PBMCsis positively associated with the expected clinical benefit of PD-1blockade treatment of the subject, wherein the expression level ofIL11RA on CD8⁺ T cells in the PBMCs is positively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject,wherein the expression level of MLL on CD8⁺ T cells in the PBMCs ispositively associated with the expected clinical benefit of PD-1blockade treatment of the subject, wherein the expression level ofHSPBAP1 on CD8⁺ T cells in the PBMCs is positively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject,wherein the expression level of MAP4K4 on CD8⁺ T cells in the PBMCs ispositively associated with the expected clinical benefit of PD-1blockade treatment of the subject, wherein the expression level of HELLSon CD8⁺ T cells in the PBMCs is positively associated with the expectedclinical benefit of PD-1 blockade treatment of the subject, wherein theexpression level of C1QTNF3 on CD8⁺ T cells in the PBMCs is positivelyassociated with the expected clinical benefit of PD-1 blockade treatmentof the subject, wherein the expression level of UBE2D1 on CD8⁺ T cellsin the PBMCs is positively associated with the expected clinical benefitof PD-1 blockade treatment of the subject, wherein the expression levelof FAIM3 on CD8⁺ T cells in the PBMCs is positively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject,wherein the expression level of IFI44 on CD8⁺ T cells in the PBMCs ispositively associated with the expected clinical benefit of PD-1blockade treatment of the subject, wherein the expression level of H2BFMon CD8⁺ T cells in the PBMCs is positively associated with the expectedclinical benefit of PD-1 blockade treatment of the subject, wherein theexpression level of GATAD1 on CD8^(|) T cells in the PBMCs is positivelyassociated with the expected clinical benefit of PD-1 blockade treatmentof the subject, wherein the expression level of CASP8 on CD8⁺ T cells inthe PBMCs is positively associated with the expected clinical benefit ofPD-1 blockade treatment of the subject, wherein the expression level ofSOCS1 on CD8⁺ T cells in the PBMCs is negatively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject,wherein the expression level of PDCD6 on CD8⁺ T cells in the PBMCs isnegatively associated with the expected clinical benefit of PD-1blockade treatment of the subject, wherein the expression level ofNY-ESO-1 on CD8⁺ T cells in the PBMCs is negatively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject,wherein the expression level of MART-1 on CD8⁺ T cells in the PBMCs isnegatively associated with the expected clinical benefit of PD-1blockade treatment of the subject, wherein an increase in the expressionlevel of LASS6 on CD8⁺ T cells in the PBMCs following PD-1 blockadetreatment is positively associated with the expected clinical benefit ofPD-1 blockade treatment of the subject, wherein a decrease in theexpression level of GADD45B on CD8⁺ T cells in the PBMCs following PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein a decrease inthe expression level of EGR2 on CD8⁺ T cells in the PBMCs following PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein a decrease inthe expression level of EGR1 on CD8⁺ T cells in the PBMCs following PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein a decrease inthe expression level of GADD45G on CD8⁺ T cells in the PBMCs followingPD-1 blockade treatment is positively associated with the expectedclinical benefit of PD-1 blockade treatment of the subject, wherein adecrease in the expression level of CKS2 on CD8^(|) T cells in the PBMCsfollowing PD-1 blockade treatment is positively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject,wherein a decrease in the expression level of CDKN1C on CD8⁺ T cells inthe PBMCs following PD-1 blockade treatment is positively associatedwith the expected clinical benefit of PD-1 blockade treatment of thesubject, wherein a decrease in the expression level of IER5 on CD8⁺ Tcells in the PBMCs following PD-1 blockade treatment is positivelyassociated with the expected clinical benefit of PD-1 blockade treatmentof the subject, wherein a decrease in the expression level of ING1 onCD8⁺ T cells in the PBMCs following PD-1 blockade treatment ispositively associated with the expected clinical benefit of PD-1blockade treatment of the subject, wherein a decrease in the expressionlevel of IFIH1 on CD8⁺ T cells in the PBMCs following PD-1 blockadetreatment is positively associated with the expected clinical benefit ofPD-1 blockade treatment of the subject, wherein a decrease in theexpression level of IFNG on CD8⁺ T cells in the PBMCs following PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, and wherein adecrease in the expression level of IER5L on CD8⁺ T cells in the PBMCsfollowing PD-1 blockade treatment is positively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject,wherein an increase in the expression level of NY-ESO-1 on CD8⁺ T cellsin the PBMCs following PD-1 blockade treatment is positively associatedwith the expected clinical benefit of PD-1 blockade treatment of thesubject, wherein an increase in the expression level of MART-1 on CD8⁺ Tcells in the PBMCs following PD-1 blockade treatment is positivelyassociated with the expected clinical benefit of PD-1 blockade treatmentof the subject, wherein a decrease in the number or frequency of Tregulatory cells (Tregs) in CD4⁺ T cells in PBMCs following PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein an increasein the number or frequency of Tregs cells in CD4⁺ T cells in PBMCsfollowing PD-1 blockade treatment is negatively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject,wherein a decrease in the number or frequency of CTLA-4⁺CD4⁺ cells inCD4⁺ T cells in PBMCs following PD-1 blockade treatment is positivelyassociated with the expected clinical benefit of PD-1 blockade treatmentof the subject, wherein an increase in the number or frequency ofCTLA-4⁺CD4⁺ cells in CD4⁺ T cells in PBMCs following PD-1 blockadetreatment is negatively associated with the expected clinical benefit ofPD-1 blockade treatment of the subject, wherein a decrease in the numberor frequency of CTLA-4⁺CD8⁺ cells in CD8^(|) T cells in PBMCs followingPD-1 blockade treatment is positively associated with the expectedclinical benefit of PD-1 blockade treatment of the subject, and whereinan increase in the number or frequency of CTLA-4⁺CD8⁺ cells in CD8⁺ Tcells in PBMCs following PD-1 blockade treatment is negativelyassociated with the expected clinical benefit of PD-1 blockade treatmentof the subject.
 2. The method of claim 1, wherein the PBMCs are assayedprior to PD-1 blockade treatment of the subject.
 3. The method of claim2, wherein the expression level of PTEN on CD8⁺ T cells in the PBMCsprior to PD-1 blockade treatment is positively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject,wherein the expression level of GATAD1 on CD8⁺ T cells in the PBMCsprior to PD-1 blockade treatment is positively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject,wherein the expression level of NKTR on CD8⁺ T cells in the PBMCs priorto PD-1 blockade treatment is positively associated with the expectedclinical benefit of PD-1 blockade treatment of the subject, wherein theexpression level of CD276 on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is negatively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein theexpression level of NSUN6 on CD8^(|) T cells in the PBMCs prior to PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein theexpression level of CCAR1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein theexpression level of SMYD2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein theexpression level of FBXO9 on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein theexpression level of NCRNA00153 on CD8⁺ T cells in the PBMCs prior toPD-1 blockade treatment is positively associated with the expectedclinical benefit of PD-1 blockade treatment of the subject, wherein theexpression level of NSMAF on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein theexpression level of P2RY4 on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is negatively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein theexpression level of RCOR2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is negatively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein theexpression level of NUB1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is negatively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein theexpression level of FCRL2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is negatively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein theexpression level of NUCKS1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is negatively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein theexpression level of SHC2 on CD8^(|) T cells in the PBMCs prior to PD-1blockade treatment is negatively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein theexpression level of SCD5 on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is negatively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein theexpression level of USP9Y on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein theexpression level of HSPD1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein theexpression level of CISH on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein theexpression level of KLF12 on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein theexpression level of NCF4 on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein theexpression level of SFRS7 on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein theexpression level of CDC14A on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein theexpression level of MAP2K5 on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein theexpression level of IL11RA on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein theexpression level of MLL on CD8^(|) T cells in the PBMCs prior to PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein theexpression level of HSPBAP1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein theexpression level of MAP4K4 on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein theexpression level of HELLS on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein theexpression level of C1QTNF3 on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein theexpression level of UBE2D1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein theexpression level of FAIM3 on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein theexpression level of IFI44 on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein theexpression level of H2BFM on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein theexpression level of GATAD1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein theexpression level of CASP8 on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein theexpression level of SOCS1 on CD8^(|) T cells in the PBMCs prior to PD-1blockade treatment is negatively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein theexpression level of PDCD6 on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is negatively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein theexpression level of NY-ESO-1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is negatively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, and wherein theexpression level of MART-1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is negatively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject.
 4. The method of anyone of claims 1 to 3, wherein the expression prior to PD-1 blockadetreatment is compared to a reference level of expression, wherein anexpression level of PTEN on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment greater than the reference level of PTEN indicatesthat the subject can expect a clinical benefit from PD-1 blockadetreatment, wherein an expression level of GATAD1 on CD8⁺ T cells in thePBMCs prior to PD-1 blockade greater than the reference level of GATAD1indicates that the subject can expect a clinical benefit from PD-1blockade treatment, wherein an expression level of NKTR on CD8⁺ T cellsin the PBMCs prior to PD-1 blockade greater than the reference level ofNKTR indicates that the subject can expect a clinical benefit from PD-1blockade treatment, wherein an expression level of CD276 on CD8⁺ T cellsin the PBMCs prior to PD-1 blockade lower than the reference level ofCD276 indicates that the subject can expect a clinical benefit from PD-1blockade treatment, wherein an expression level of NSUN6 on CD8⁺ T cellsin the PBMCs prior to PD-1 blockade greater than the reference level ofNSUN6 indicates that the subject can expect a clinical benefit from PD-1blockade treatment, wherein an expression level of CCAR1 on CD8⁺ T cellsin the PBMCs prior to PD-1 blockade greater than the reference level ofCCAR1 indicates that the subject can expect a clinical benefit from PD-1blockade treatment, wherein an expression level of SMYD2 on CD8^(|) Tcells in the PBMCs prior to PD-1 blockade greater than the referencelevel of SMYD2 indicates that the subject can expect a clinical benefitfrom PD-1 blockade treatment, wherein an expression level of FBXO9 onCD8⁺ T cells in the PBMCs prior to PD-1 blockade greater than thereference level of FBXO9 indicates that the subject can expect aclinical benefit from PD-1 blockade treatment, wherein an expressionlevel of NCRNA00153 on CD8⁺ T cells in the PBMCs prior to PD-1 blockadegreater than the reference level of NCRNA00153 indicates that thesubject can expect a clinical benefit from PD-1 blockade treatment,wherein an expression level of NSMAF on CD8^(|) T cells in the PBMCsprior to PD-1 blockade greater than the reference level of NSMAFindicates that the subject can expect a clinical benefit from PD-1blockade treatment, wherein an expression level of P2RY4 on CD8⁺ T cellsin the PBMCs prior to PD-1 blockade lower than the reference level ofP2RY4 indicates that the subject can expect a clinical benefit from PD-1blockade treatment, wherein an expression level of RCOR2 on CD8⁺ T cellsin the PBMCs prior to PD-1 blockade lower than the reference level ofRCOR2 indicates that the subject can expect a clinical benefit from PD-1blockade treatment, wherein an expression level of NUB1 on CD8⁺ T cellsin the PBMCs prior to PD-1 blockade lower than the reference level ofNUB1 indicates that the subject can expect a clinical benefit from PD-1blockade treatment, wherein an expression level of FCRL2 on CD8⁺ T cellsin the PBMCs prior to PD-1 blockade lower than the reference level ofFCRL2 indicates that the subject can expect a clinical benefit from PD-1blockade treatment, wherein an expression level of NUCKS1 on CD8⁺ Tcells in the PBMCs prior to PD-1 blockade lower than the reference levelof NUCKS1 indicates that the subject can expect a clinical benefit fromPD-1 blockade treatment, wherein an expression level of SHC2 on CD8⁺ Tcells in the PBMCs prior to PD-1 blockade lower than the reference levelof SHC2 indicates that the subject can expect a clinical benefit fromPD-1 blockade treatment, wherein an expression level of SCD5 on CD8^(|)T cells in the PBMCs prior to PD-1 blockade lower than the referencelevel of SCD5 indicates that the subject can expect a clinical benefitfrom PD-1 blockade treatment, wherein an expression level of USP9Y onCD8⁺ T cells in the PBMCs prior to PD-1 blockade greater than thereference level of USP9Y indicates that the subject can expect aclinical benefit from PD-1 blockade treatment, wherein an expressionlevel of HSPD1 on CD8⁺ T cells in the PBMCs prior to PD-1 blockadegreater than the reference level of HSPD1 indicates that the subject canexpect a clinical benefit from PD-1 blockade treatment, wherein anexpression level of CISH on CD8⁺ T cells in the PBMCs prior to PD-1blockade greater than the reference level of CISH indicates that thesubject can expect a clinical benefit from PD-1 blockade treatment,wherein an expression level of KLF2 on CD8⁺ T cells in the PBMCs priorto PD-1 blockade greater than the reference level of KLF12 indicatesthat the subject can expect a clinical benefit from PD-1 blockadetreatment, wherein an expression level of NCF4 on CD8⁺ T cells in thePBMCs prior to PD-1 blockade greater than the reference level of NCF4indicates that the subject can expect a clinical benefit from PD-1blockade treatment, wherein an expression level of SFRS7 on CD8⁺ T cellsin the PBMCs prior to PD-1 blockade greater than the reference level ofSFRS7 indicates that the subject can expect a clinical benefit from PD-1blockade treatment, wherein an expression level of CDC14A on CD8⁺ Tcells in the PBMCs prior to PD-1 blockade greater than the referencelevel of CDC14A indicates that the subject can expect a clinical benefitfrom PD-1 blockade treatment, wherein an expression level of MAP2K5 onCD8⁺ T cells in the PBMCs prior to PD-1 blockade greater than thereference level of MAP2K5 indicates that the subject can expect aclinical benefit from PD-1 blockade treatment, wherein an expressionlevel of IL11RA on CD8⁺ T cells in the PBMCs prior to PD-1 blockadegreater than the reference level of IL11RA indicates that the subjectcan expect a clinical benefit from PD-1 blockade treatment, wherein anexpression level of MLL on CD8⁺ T cells in the PBMCs prior to PD-1blockade greater than the reference level of MLL indicates that thesubject can expect a clinical benefit from PD-1 blockade treatment,wherein an expression level of HSPBAP1 on CD8^(|) T cells in the PBMCsprior to PD-1 blockade greater than the reference level of HSPBAP1indicates that the subject can expect a clinical benefit from PD-1blockade treatment, wherein an expression level of MAP4K4 on CD8⁺ Tcells in the PBMCs prior to PD-1 blockade greater than the referencelevel of MAP4K4 indicates that the subject can expect a clinical benefitfrom PD-1 blockade treatment, wherein an expression level of HELLS onCD8⁺ T cells in the PBMCs prior to PD-1 blockade greater than thereference level of HELLS indicates that the subject can expect aclinical benefit from PD-1 blockade treatment, wherein an expressionlevel of C1QTNF3 on CD8⁺ T cells in the PBMCs prior to PD-1 blockadegreater than the reference level of C1QTNF3 indicates that the subjectcan expect a clinical benefit from PD-1 blockade treatment, wherein anexpression level of UBE2D1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade greater than the reference level of UBE2D1 indicates that thesubject can expect a clinical benefit from PD-1 blockade treatment,wherein an expression level of FAIM3 on CD8⁺ T cells in the PBMCs priorto PD-1 blockade greater than the reference level of FAIM3 indicatesthat the subject can expect a clinical benefit from PD-1 blockadetreatment, wherein an expression level of IFI44 on CD8⁺ T cells in thePBMCs prior to PD-1 blockade greater than the reference level of IFI44indicates that the subject can expect a clinical benefit from PD-1blockade treatment, wherein an expression level of H2BFM on CD8⁺ T cellsin the PBMCs prior to PD-1 blockade greater than the reference level ofH2BFM indicates that the subject can expect a clinical benefit from PD-1blockade treatment, wherein an expression level of GATAD1 on CD8⁺ Tcells in the PBMCs prior to PD-1 blockade greater than the referencelevel of GATAD1 indicates that the subject can expect a clinical benefitfrom PD-1 blockade treatment, wherein an expression level of CASP8 onCD8⁺ T cells in the PBMCs prior to PD-1 blockade greater than thereference level of CASP8 indicates that the subject can expect aclinical benefit from PD-1 blockade treatment, wherein an expressionlevel of SOCS1 on CD8⁺ T cells in the PBMCs prior to PD-1 blockade lowerthan the reference level of SOCS1 indicates that the subject can expecta clinical benefit from PD-1 blockade treatment, and wherein anexpression level of PDCD6 on CD8^(|) T cells in the PBMCs prior to PD-1blockade lower than the reference level of PDCD6 indicates that thesubject can expect a clinical benefit from PD-1 blockade treatment. 5.The method of any one of claims 1 to 3, wherein the expression prior toPD-1 blockade treatment is compared to a reference level of expression,wherein an expression level of PTEN on CD8⁺ T cells in the PBMCs priorto PD-1 blockade treatment more than 1.2 times the reference level ofPTEN indicates that the subject can expect a clinical benefit from PD-1blockade treatment, wherein an expression level of GATAD1 on CD8^(|) Tcells in the PBMCs prior to PD-1 blockade more than 1.1 times thereference level of GATAD1 indicates that the subject can expect aclinical benefit from PD-1 blockade treatment, wherein an expressionlevel of NKTR on CD8⁺ T cells in the PBMCs prior to PD-1 blockade morethan 1.1 times the reference level of NKTR indicates that the subjectcan expect a clinical benefit from PD-1 blockade treatment, wherein anexpression level of CD276 on CD8⁺ T cells in the PBMCs prior to PD-1blockade less than 0.9 times the reference level of CD276 indicates thatthe subject can expect a clinical benefit from PD-1 blockade treatment,wherein an expression level of NSUN6 on CD8⁺ T cells in the PBMCs priorto PD-1 blockade more than 1.4 times the reference level of NSUN6indicates that the subject can expect a clinical benefit from PD-1blockade treatment, wherein an expression level of CCAR1 on CD8⁺ T cellsin the PBMCs prior to PD-1 blockade more than 1.2 times the referencelevel of CCAR1 indicates that the subject can expect a clinical benefitfrom PD-1 blockade treatment, wherein an expression level of SMYD2 onCD8⁺ T cells in the PBMCs prior to PD-1 blockade more than 1.2 times thereference level of SMYD2 indicates that the subject can expect aclinical benefit from PD-1 blockade treatment, wherein an expressionlevel of FBXO9 on CD8^(|) T cells in the PBMCs prior to PD-1 blockademore than 1.2 times the reference level of FBXO9 indicates that thesubject can expect a clinical benefit from PD-1 blockade treatment,wherein an expression level of NCRNA00153 on CD8⁺ T cells in the PBMCsprior to PD-1 blockade more than 1.2 times the reference level ofNCRNA00153 indicates that the subject can expect a clinical benefit fromPD-1 blockade treatment, wherein an expression level of NSMAF on CD8⁺ Tcells in the PBMCs prior to PD-1 blockade more than 1.2 times thereference level of NSMAF indicates that the subject can expect aclinical benefit from PD-1 blockade treatment, wherein an expressionlevel of P2RY4 on CD8⁺ T cells in the PBMCs prior to PD-1 blockade lessthan 0.9 times the reference level of P2RY4 indicates that the subjectcan expect a clinical benefit from PD-1 blockade treatment, wherein anexpression level of RCOR2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade less than 0.9 times the reference level of RCOR2 indicates thatthe subject can expect a clinical benefit from PD-1 blockade treatment,wherein an expression level of NUB1 on CD8⁺ T cells in the PBMCs priorto PD-1 blockade less than 0.9 times the reference level of NUB1indicates that the subject can expect a clinical benefit from PD-1blockade treatment, wherein an expression level of FCRL2 on CD8⁺ T cellsin the PBMCs prior to PD-1 blockade less than 0.9 times the referencelevel of FCRL2 indicates that the subject can expect a clinical benefitfrom PD-1 blockade treatment, wherein an expression level of NUCKS1 onCD8⁺ T cells in the PBMCs prior to PD-1 blockade less than 0.8 times thereference level of NUCKS1 indicates that the subject can expect aclinical benefit from PD-1 blockade treatment, wherein an expressionlevel of SHC2 on CD8⁺ T cells in the PBMCs prior to PD-1 blockade lessthan 0.8 times the reference level of SHC2 indicates that the subjectcan expect a clinical benefit from PD-1 blockade treatment, wherein anexpression level of SCD5 on CD8⁺ T cells in the PBMCs prior to PD-1blockade less than 0.8 times the reference level of SCD5 indicates thatthe subject can expect a clinical benefit from PD-1 blockade treatment,wherein an expression level of USP9Y on CD8⁺ T cells in the PBMCs priorto PD-1 blockade more than 1.6 times the reference level of USP9Yindicates that the subject can expect a clinical benefit from PD-1blockade treatment, wherein an expression level of HSPD1 on CD8⁺ T cellsin the PBMCs prior to PD-1 blockade more than 1.5 times the referencelevel of HSPD1 indicates that the subject can expect a clinical benefitfrom PD-1 blockade treatment, wherein an expression level of CISH onCD8⁺ T cells in the PBMCs prior to PD-1 blockade more than 1.4 times thereference level of CISH indicates that the subject can expect a clinicalbenefit from PD-1 blockade treatment, wherein an expression level ofKLF2 on CD8⁺ T cells in the PBMCs prior to PD-1 blockade more than 1.3times the reference level of KLF12 indicates that the subject can expecta clinical benefit from PD-1 blockade treatment, wherein an expressionlevel of NCF4 on CD8⁺ T cells in the PBMCs prior to PD-1 blockade morethan 1.3 times the reference level of NCF4 indicates that the subjectcan expect a clinical benefit from PD-1 blockade treatment, wherein anexpression level of SFRS7 on CD8⁺ T cells in the PBMCs prior to PD-1blockade more than 1.2 times the reference level of SFRS7 indicates thatthe subject can expect a clinical benefit from PD-1 blockade treatment,wherein an expression level of CDC14A on CD8⁺ T cells in the PBMCs priorto PD-1 blockade more than 1.2 times the reference level of CDC14Aindicates that the subject can expect a clinical benefit from PD-1blockade treatment, wherein an expression level of MAP2K5 on CD8⁺ Tcells in the PBMCs prior to PD-1 blockade more than 1.2 times thereference level of MAP2K5 indicates that the subject can expect aclinical benefit from PD-1 blockade treatment, wherein an expressionlevel of IL11RA on CD8⁺ T cells in the PBMCs prior to PD-1 blockade morethan 1.2 times the reference level of IL11RA indicates that the subjectcan expect a clinical benefit from PD-1 blockade treatment, wherein anexpression level of MLL on CD8⁺ T cells in the PBMCs prior to PD-1blockade more than 1.2 times the reference level of MLL indicates thatthe subject can expect a clinical benefit from PD-1 blockade treatment,wherein an expression level of HSPBAP1 on CD8^(|) T cells in the PBMCsprior to PD-1 blockade more than 1.2 times the reference level ofHSPBAP1 indicates that the subject can expect a clinical benefit fromPD-1 blockade treatment, wherein an expression level of MAP4K4 on CD8⁺ Tcells in the PBMCs prior to PD-1 blockade more than 1.2 times thereference level of MAP4K4 indicates that the subject can expect aclinical benefit from PD-1 blockade treatment, wherein an expressionlevel of HELLS on CD8⁺ T cells in the PBMCs prior to PD-1 blockade morethan 1.2 times the reference level of HELLS indicates that the subjectcan expect a clinical benefit from PD-1 blockade treatment, wherein anexpression level of C1QTNF3 on CD8^(|) T cells in the PBMCs prior toPD-1 blockade more than 1.2 times the reference level of C1QTNF3indicates that the subject can expect a clinical benefit from PD-1blockade treatment, wherein an expression level of UBE2D1 on CD8⁺ Tcells in the PBMCs prior to PD-1 blockade more than 1.2 times thereference level of UBE2D1 indicates that the subject can expect aclinical benefit from PD-1 blockade treatment, wherein an expressionlevel of FAIM3 on CD8⁺ T cells in the PBMCs prior to PD-1 blockade morethan 1.2 times the reference level of FAIM3 indicates that the subjectcan expect a clinical benefit from PD-1 blockade treatment, wherein anexpression level of IFI44 on CD8⁺ T cells in the PBMCs prior to PD-1blockade more than 1.2 times the reference level of IFI44 indicates thatthe subject can expect a clinical benefit from PD-1 blockade treatment,wherein an expression level of H2BFM on CD8⁺ T cells in the PBMCs priorto PD-1 blockade more than 1.2 times the reference level of H2BFMindicates that the subject can expect a clinical benefit from PD-1blockade treatment, wherein an expression level of GATAD1 on CD8⁺ Tcells in the PBMCs prior to PD-1 blockade more than 1.1 times thereference level of GATAD1 indicates that the subject can expect aclinical benefit from PD-1 blockade treatment, wherein an expressionlevel of CASP8 on CD8⁺ T cells in the PBMCs prior to PD-1 blockade morethan 1.1 times the reference level of CASP8 indicates that the subjectcan expect a clinical benefit from PD-1 blockade treatment, wherein anexpression level of SOCS1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade less than 0.9 times the reference level of SOCS1 indicates thatthe subject can expect a clinical benefit from PD-1 blockade treatment,and wherein an expression level of PDCD6 on CD8⁺ T cells in the PBMCsprior to PD-1 blockade less than 0.9 times the reference level of PDCD6indicates that the subject can expect a clinical benefit from PD-1blockade treatment.
 6. The method of any one of claims 1 to 5 furthercomprising treating the subject with a PD-1 blockade treatment if theexpression level of PTEN on CD8⁺ T cells in the PBMCs prior to PD-1blockade treatment is more than 1.2 times the reference level of PTEN,the expression level of GATAD1 on CD8⁺ T cells in the PBMCs prior toPD-1 blockade is more than 1.1 times the reference level of GATAD1, theexpression level of NKTR on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.1 times the reference level of NKTR, theexpression level of CD276 on CD8^(|) T cells in the PBMCs prior to PD-1blockade is less than 0.9 times the reference level of CD276, theexpression level of NSUN6 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.4 times the reference level of NSUN6, theexpression level of CCAR1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.2 times the reference level of CCAR1, theexpression level of SMYD2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.2 times the reference level of SMYD2, theexpression level of FBXO9 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.2 times the reference level of FBXO9, theexpression level of NCRNA00153 on CD8^(|) T cells in the PBMCs prior toPD-1 blockade is more than 1.2 times the reference level of NCRNA00153,the expression level of NSMAF on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.2 times the reference level of NSMAF, theexpression level of P2RY4 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 0.9 times the reference level of P2RY4, theexpression level of RCOR2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 0.9 times the reference level of RCOR2, theexpression level of NUB1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 0.9 times the reference level of NUB1, theexpression level of FCRL2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 0.9 times the reference level of FCRL2, theexpression level of NUCKS1 on CD8^(|) T cells in the PBMCs prior to PD-1blockade is less than 0.8 times the reference level of NUCKS1, theexpression level of SHC2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 0.8 times the reference level of SHC2, theexpression level of SCD5 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 0.8 times the reference level of SCD5, thefrequency of NY-ESO-1⁺CD8⁺ cells in CD8⁺ T cells in the PBMCs prior toPD-1 blockade treatment is less than 0.5%, wherein the frequency ofMART-1⁺CD8⁺ cells in CD8⁺ T cells in the PBMCs prior to PD-1 blockadetreatment is less than 0.5%, or a combination.
 7. The method of any oneof claims 1 to 5 further comprising treating the subject with atreatment other than a PD-1 blockade treatment if the expression levelof PTEN on CD8⁺ T cells in the PBMCs prior to PD-1 blockade treatment isless than 1.1 times the reference level of PTEN, the expression level ofGATAD1 on CD8⁺ T cells in the PBMCs prior to PD-1 blockade is less than1.0 times the reference level of GATAD1, the expression level of NKTR onCD8⁺ T cells in the PBMCs prior to PD-1 blockade is less than 1.0 timesthe reference level of NKTR, the expression level of CD276 on CD8^(|) Tcells in the PBMCs prior to PD-1 blockade is more than 1.0 times thereference level of CD276, the expression level of NSUN6 on CD8⁺ T cellsin the PBMCs prior to PD-1 blockade is less than 1.2 times the referencelevel of NSUN6, the expression level of CCAR1 on CD8⁺ T cells in thePBMCs prior to PD-1 blockade is less than 1.1 times the reference levelof CCAR1, the expression level of SMYD2 on CD8⁺ T cells in the PBMCsprior to PD-1 blockade is less than 1.1 times the reference level ofSMYD2, the expression level of FBXO9 on CD8⁺ T cells in the PBMCs priorto PD-1 blockade is less than 1.1 times the reference level of FBXO9,the expression level of NCRNA00153 on CD8⁺ T cells in the PBMCs prior toPD-1 blockade is less than 1.1 times the reference level of NCRNA00153,the expression level of NSMAF on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 1.1 times the reference level of NSMAF, theexpression level of P2RY4 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.0 times the reference level of P2RY4, theexpression level of RCOR2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.0 times the reference level of RCOR2, theexpression level of NUB1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.0 times the reference level of NUB1, theexpression level of FCRL2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.0 times the reference level of FCRL2, theexpression level of NUCKS1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 0.9 times the reference level of NUCKS1, theexpression level of SHC2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 0.9 times the reference level of SHC2, theexpression level of SCD5 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 0.9 times the reference level of SCD5, thefrequency of NY-ESO-1⁺CD8⁺ cells in CD8⁺ T cells in the PBMCs prior toPD-1 blockade treatment is more than 0.5%, the frequency of MART-1⁺CD8⁺cells in CD8⁺ T cells in the PBMCs prior to PD-1 blockade treatment ismore than 0.5%, or a combination.
 8. The method of any one of claims 1to 5 further comprising treating the subject with both a PD-1 blockadetreatment and a different treatment if the expression level of PTEN onCD8⁺ T cells in the PBMCs prior to PD-1 blockade treatment is less than1.1 times the reference level of PTEN, the expression level of GATAD1 onCD8⁺ T cells in the PBMCs prior to PD-1 blockade is less than 1.0 timesthe reference level of GATAD1, the expression level of NKTR on CD8⁺ Tcells in the PBMCs prior to PD-1 blockade is less than 1.0 times thereference level of NKTR, the expression level of CD276 on CD8⁺ T cellsin the PBMCs prior to PD-1 blockade is more than 1.0 times the referencelevel of CD276, the expression level of NSUN6 on CD8^(|) T cells in thePBMCs prior to PD-1 blockade is less than 1.2 times the reference levelof NSUN6, the expression level of CCAR1 on CD8⁺ T cells in the PBMCsprior to PD-1 blockade is less than 1.1 times the reference level ofCCAR1, the expression level of SMYD2 on CD8⁺ T cells in the PBMCs priorto PD-1 blockade is less than 1.1 times the reference level of SMYD2,the expression level of FBXO9 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 1.1 times the reference level of FBXO9, theexpression level of NCRNA00153 on CD8⁺ T cells in the PBMCs prior toPD-1 blockade is less than 1.1 times the reference level of NCRNA00153,the expression level of NSMAF on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 1.1 times the reference level of NSMAF, theexpression level of P2RY4 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.0 times the reference level of P2RY4, theexpression level of RCOR2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.0 times the reference level of RCOR2, theexpression level of NUB1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.0 times the reference level of NUB1, theexpression level of FCRL2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.0 times the reference level of FCRL2, theexpression level of NUCKS1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 0.9 times the reference level of NUCKS1, theexpression level of SHC2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 0.9 times the reference level of SHC2, theexpression level of SCD5 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 0.9 times the reference level of SCD5, thefrequency of NY-ESO-1⁺CD8⁺ cells in CD8⁺ T cells in the PBMCs prior toPD-1 blockade treatment is more than 0.5%, the frequency of MART-1⁺CD8⁺cells in CD8⁺ T cells in the PBMCs prior to PD-1 blockade treatment ismore than 0.5%, or a combination.
 9. The method of any one of claims 1to 5 further comprising selecting a subject for PD-1 blockade treatmentif the expression level of PTEN on CD8⁺ T cells in the PBMCs prior toPD-1 blockade treatment is more than 1.2 times the reference level ofPTEN, the expression level of GATAD1 on CD8⁺ T cells in the PBMCs priorto PD-1 blockade is more than 1.1 times the reference level of GATAD1,the expression level of NKTR on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.1 times the reference level of NKTR, theexpression level of CD276 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 0.9 times the reference level of CD276, theexpression level of NSUN6 on CD8^(|) T cells in the PBMCs prior to PD-1blockade is more than 1.4 times the reference level of NSUN6, theexpression level of CCAR1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.2 times the reference level of CCAR1, theexpression level of SMYD2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.2 times the reference level of SMYD2, theexpression level of FBXO9 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.2 times the reference level of FBXO9, theexpression level of NCRNA00153 on CD8⁺ T cells in the PBMCs prior toPD-1 blockade is more than 1.2 times the reference level of NCRNA00153,the expression level of NSMAF on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.2 times the reference level of NSMAF, theexpression level of P2RY4 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 0.9 times the reference level of P2RY4, theexpression level of RCOR2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 0.9 times the reference level of RCOR2, theexpression level of NUB1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 0.9 times the reference level of NUB1, theexpression level of FCRL2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 0.9 times the reference level of FCRL2, theexpression level of NUCKS1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 0.8 times the reference level of NUCKS1, theexpression level of SHC2 on CD8^(|) T cells in the PBMCs prior to PD-1blockade is less than 0.8 times the reference level of SHC2, theexpression level of SCD5 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 0.8 times the reference level of SCD5, thefrequency of NY-ESO-1 CD8⁺ cells in CD8⁺ T cells in the PBMCs prior toPD-1 blockade treatment is less than 0.5%, the frequency of MART-1⁺CD8⁺cells in CD8⁺ T cells in the PBMCs prior to PD-1 blockade treatment isless than 0.5%, or a combination.
 10. The method of any one of claims 1to 5 further comprising selecting a subject for a treatment other than aPD-1 blockade treatment if the expression level of PTEN on CD8⁺ T cellsin the PBMCs prior to PD-1 blockade treatment is less than 1.1 times thereference level of PTEN, the expression level of GATAD1 on CD8⁺ T cellsin the PBMCs prior to PD-1 blockade is less than 1.0 times the referencelevel of GATAD1, the expression level of NKTR on CD8⁺ T cells in thePBMCs prior to PD-1 blockade is less than 1.0 times the reference levelof NKTR, the expression level of CD276 on CD8⁺ T cells in the PBMCsprior to PD-1 blockade is more than 1.0 times the reference level ofCD276, the expression level of NSUN6 on CD8^(|) T cells in the PBMCsprior to PD-1 blockade is less than 1.2 times the reference level ofNSUN6, the expression level of CCAR1 on CD8⁺ T cells in the PBMCs priorto PD-1 blockade is less than 1.1 times the reference level of CCAR1,the expression level of SMYD2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 1.1 times the reference level of SMYD2, theexpression level of FBXO9 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 1.1 times the reference level of FBXO9, theexpression level of NCRNA00153 on CD8⁺ T cells in the PBMCs prior toPD-1 blockade is less than 1.1 times the reference level of NCRNA00153,the expression level of NSMAF on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 1.1 times the reference level of NSMAF, theexpression level of P2RY4 on CD8^(|) T cells in the PBMCs prior to PD-1blockade is more than 1.0 times the reference level of P2RY4, theexpression level of RCOR2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.0 times the reference level of RCOR2, theexpression level of NUB1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.0 times the reference level of NUB1, theexpression level of FCRL2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.0 times the reference level of FCRL2, theexpression level of NUCKS1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 0.9 times the reference level of NUCKS1, theexpression level of SHC2 on CD8^(|) T cells in the PBMCs prior to PD-1blockade is more than 0.9 times the reference level of SHC2, theexpression level of SCD5 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 0.9 times the reference level of SCD5, thefrequency of NY-ESO-1⁺CD8⁺ cells in CD8⁺ T cells in the PBMCs prior toPD-1 blockade treatment is more than 0.5%, the frequency of MART-1⁺CD8⁺cells in CD8⁺ T cells in the PBMCs prior to PD-1 blockade treatment ismore than 0.5%, or a combination.
 11. The method of any one of claims 1to 5 further comprising selecting a subject for treatment with both aPD-1 blockade treatment and a different treatment if the expressionlevel of PTEN on CD8⁺ T cells in the PBMCs prior to PD-1 blockadetreatment is less than 1.1 times the reference level of PTEN, theexpression level of GATAD1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 1.0 times the reference level of GATAD1, theexpression level of NKTR on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 1.0 times the reference level of NKTR, theexpression level of CD276 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.0 times the reference level of CD276, theexpression level of NSUN6 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 1.2 times the reference level of NSUN6, theexpression level of CCAR1 on CD8^(|) T cells in the PBMCs prior to PD-1blockade is less than 1.1 times the reference level of CCAR1, theexpression level of SMYD2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 1.1 times the reference level of SMYD2, theexpression level of FBXO9 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 1.1 times the reference level of FBXO9, theexpression level of NCRNA00153 on CD8⁺ T cells in the PBMCs prior toPD-1 blockade is less than 1.1 times the reference level of NCRNA00153,the expression level of NSMAF on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 1.1 times the reference level of NSMAF, theexpression level of P2RY4 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.0 times the reference level of P2RY4, theexpression level of RCOR2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.0 times the reference level of RCOR2, theexpression level of NUB1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.0 times the reference level of NUB1, theexpression level of FCRL2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.0 times the reference level of FCRL2, theexpression level of NUCKS1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 0.9 times the reference level of NUCKS1, theexpression level of SHC2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 0.9 times the reference level of SHC2, theexpression level of SCD5 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 0.9 times the reference level of SCD5, thefrequency of NY-ESO-1⁺CD8⁺ cells in CD8⁺ T cells in the PBMCs prior toPD-1 blockade treatment is more than 0.5%, the frequency of MART-1⁺CD8⁺cells in CD8⁺ T cells in the PBMCs prior to PD-1 blockade treatment ismore than 0.5%, or a combination.
 12. The method of any one of claims 1to 5, wherein the subject is treated with a PD-1 blockade treatment ifthe expression level of PTEN on CD8^(|) T cells in the PBMCs prior toPD-1 blockade treatment is more than 1.2 times the reference level ofPTEN, the expression level of GATAD1 on CD8⁺ T cells in the PBMCs priorto PD-1 blockade is more than 1.1 times the reference level of GATAD1,the expression level of NKTR on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.1 times the reference level of NKTR, theexpression level of CD276 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 0.9 times the reference level of CD276, theexpression level of NSUN6 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.4 times the reference level of NSUN6, theexpression level of CCAR1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.2 times the reference level of CCAR1, theexpression level of SMYD2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.2 times the reference level of SMYD2, theexpression level of FBXO9 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.2 times the reference level of FBXO9, theexpression level of NCRNA00153 on CD8⁺ T cells in the PBMCs prior toPD-1 blockade is more than 1.2 times the reference level of NCRNA00153,the expression level of NSMAF on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.2 times the reference level of NSMAF, theexpression level of P2RY4 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 0.9 times the reference level of P2RY4, theexpression level of RCOR2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 0.9 times the reference level of RCOR2, theexpression level of NUB1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 0.9 times the reference level of NUB1, theexpression level of FCRL2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 0.9 times the reference level of FCRL2, theexpression level of NUCKS1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 0.8 times the reference level of NUCKS1, theexpression level of SHC2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 0.8 times the reference level of SHC2, theexpression level of SCD5 on CD8^(|) T cells in the PBMCs prior to PD-1blockade is less than 0.8 times the reference level of SCD5, thefrequency of NY-ESO-1⁺CD8⁺ cells in CD8⁺ T cells in the PBMCs prior toPD-1 blockade treatment is less than 0.5%, the frequency of MART-1 CD8⁺cells in CD8⁺ T cells in the PBMCs prior to PD-1 blockade treatment isless than 0.5%, or a combination.
 13. The method of any one of claims 1to 5, wherein the subject is not treated with a PD-1 blockade treatmentif the expression level of PTEN on CD8⁺ T cells in the PBMCs prior toPD-1 blockade treatment is less than 1.1 times the reference level ofPTEN, the expression level of GATAD1 on CD8⁺ T cells in the PBMCs priorto PD-1 blockade is less than 1.0 times the reference level of GATAD1,the expression level of NKTR on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 1.0 times the reference level of NKTR, theexpression level of CD276 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.0 times the reference level of CD276, theexpression level of NSUN6 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 1.2 times the reference level of NSUN6, theexpression level of CCAR1 on CD8^(|) T cells in the PBMCs prior to PD-1blockade is less than 1.1 times the reference level of CCAR1, theexpression level of SMYD2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 1.1 times the reference level of SMYD2, theexpression level of FBXO9 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 1.1 times the reference level of FBXO9, theexpression level of NCRNA00153 on CD8⁺ T cells in the PBMCs prior toPD-1 blockade is less than 1.1 times the reference level of NCRNA00153,the expression level of NSMAF on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 1.1 times the reference level of NSMAF, theexpression level of P2RY4 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.0 times the reference level of P2RY4, theexpression level of RCOR2 on CD8^(|) T cells in the PBMCs prior to PD-1blockade is more than 1.0 times the reference level of RCOR2, theexpression level of NUB1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.0 times the reference level of NUB1, theexpression level of FCRL2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.0 times the reference level of FCRL2, theexpression level of NUCKS1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 0.9 times the reference level of NUCKS1, theexpression level of SHC2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 0.9 times the reference level of SHC2, theexpression level of SCD5 on CD8^(|) T cells in the PBMCs prior to PD-1blockade is more than 0.9 times the reference level of SCD5, thefrequency of NY-ESO-1⁺CD8⁺ cells in CD8⁺ T cells in the PBMCs prior toPD-1 blockade treatment is more than 0.5%, the frequency of MART-1⁺CD8⁺cells in CD8⁺ T cells in the PBMCs prior to PD-1 blockade treatment ismore than 0.5%, or a combination.
 14. A method of treating a subjectdiagnosed with melanoma with a PD-1 blockade treatment if the expressionlevel of PTEN on CD8⁺ T cells in the PBMCs prior to PD-1 blockadetreatment is more than 1.2 times the reference level of PTEN, theexpression level of GATAD1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.1 times the reference level of GATAD1, theexpression level of NKTR on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.1 times the reference level of NKTR, theexpression level of CD276 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 0.9 times the reference level of CD276, theexpression level of NSUN6 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.4 times the reference level of NSUN6, theexpression level of CCAR1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.2 times the reference level of CCAR1, theexpression level of SMYD2 on CD8^(|) T cells in the PBMCs prior to PD-1blockade is more than 1.2 times the reference level of SMYD2, theexpression level of FBXO9 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.2 times the reference level of FBXO9, theexpression level of NCRNA00153 on CD8⁺ T cells in the PBMCs prior toPD-1 blockade is more than 1.2 times the reference level of NCRNA00153,the expression level of NSMAF on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.2 times the reference level of NSMAF, theexpression level of P2RY4 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 0.9 times the reference level of P2RY4, theexpression level of RCOR2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 0.9 times the reference level of RCOR2, theexpression level of NUB1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 0.9 times the reference level of NUB1, theexpression level of FCRL2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 0.9 times the reference level of FCRL2, theexpression level of NUCKS1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 0.8 times the reference level of NUCKS1, theexpression level of SHC2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 0.8 times the reference level of SHC2, theexpression level of SCD5 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 0.8 times the reference level of SCD5, thefrequency of NY-ESO-1⁺CD8⁺ cells in CD8⁺ T cells in the PBMCs prior toPD-1 blockade treatment is less than 0.5%, the frequency of MART-1 CD8⁺cells in CD8⁺ T cells in the PBMCs prior to PD-1 blockade treatment isless than 0.5%, or a combination.
 15. A method of treating a subjectdiagnosed with melanoma with a treatment other than a PD-1 blockadetreatment if the expression level of PTEN on CD8⁺ T cells in the PBMCsprior to PD-1 blockade treatment is less than 1.1 times the referencelevel of PTEN, the expression level of GATAD1 on CD8^(|) T cells in thePBMCs prior to PD-1 blockade is less than 1.0 times the reference levelof GATAD1, the expression level of NKTR on CD8⁺ T cells in the PBMCsprior to PD-1 blockade is less than 1.0 times the reference level ofNKTR, the expression level of CD276 on CD8⁺ T cells in the PBMCs priorto PD-1 blockade is more than 1.0 times the reference level of CD276,the expression level of NSUN6 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 1.2 times the reference level of NSUN6, theexpression level of CCAR1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 1.1 times the reference level of CCAR1, theexpression level of SMYD2 on CD8^(|) T cells in the PBMCs prior to PD-1blockade is less than 1.1 times the reference level of SMYD2, theexpression level of FBXO9 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 1.1 times the reference level of FBXO9, theexpression level of NCRNA00153 on CD8⁺ T cells in the PBMCs prior toPD-1 blockade is less than 1.1 times the reference level of NCRNA00153,the expression level of NSMAF on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 1.1 times the reference level of NSMAF, theexpression level of P2RY4 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.0 times the reference level of P2RY4, theexpression level of RCOR2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.0 times the reference level of RCOR2, theexpression level of NUB1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.0 times the reference level of NUB1, theexpression level of FCRL2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.0 times the reference level of FCRL2, theexpression level of NUCKS1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 0.9 times the reference level of NUCKS1, theexpression level of SHC2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 0.9 times the reference level of SHC2, theexpression level of SCD5 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 0.9 times the reference level of SCD5, thefrequency of NY-ESO-1^(|)CD8^(|) cells in CD8^(|) T cells in the PBMCsprior to PD-1 blockade treatment is more than 0.5%, the frequency ofMART-1⁺CD8⁺ cells in CD8⁺ T cells in the PBMCs prior to PD-1 blockadetreatment is more than 0.5%, or a combination.
 16. A method of treatinga subject diagnosed with melanoma with both a PD-1 blockade treatmentand a different treatment if the expression level of PTEN on CD8⁺ Tcells in the PBMCs prior to PD-1 blockade treatment is less than 1.1times the reference level of PTEN, the expression level of GATAD1 onCD8⁺ T cells in the PBMCs prior to PD-1 blockade is less than 1.0 timesthe reference level of GATAD1, the expression level of NKTR on CD8⁺ Tcells in the PBMCs prior to PD-1 blockade is less than 1.0 times thereference level of NKTR, the expression level of CD276 on CD8⁺ T cellsin the PBMCs prior to PD-1 blockade is more than 1.0 times the referencelevel of CD276, the expression level of NSUN6 on CD8⁺ T cells in thePBMCs prior to PD-1 blockade is less than 1.2 times the reference levelof NSUN6, the expression level of CCAR1 on CD8⁺ T cells in the PBMCsprior to PD-1 blockade is less than 1.1 times the reference level ofCCAR1, the expression level of SMYD2 on CD8^(|) T cells in the PBMCsprior to PD-1 blockade is less than 1.1 times the reference level ofSMYD2, the expression level of FBXO9 on CD8⁺ T cells in the PBMCs priorto PD-1 blockade is less than 1.1 times the reference level of FBXO9,the expression level of NCRNA00153 on CD8⁺ T cells in the PBMCs prior toPD-1 blockade is less than 1.1 times the reference level of NCRNA00153,the expression level of NSMAF on CD8⁺ T cells in the PBMCs prior to PD-1blockade is less than 1.1 times the reference level of NSMAF, theexpression level of P2RY4 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.0 times the reference level of P2RY4, theexpression level of RCOR2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.0 times the reference level of RCOR2, theexpression level of NUB1 on CD8^(|) T cells in the PBMCs prior to PD-1blockade is more than 1.0 times the reference level of NUB1, theexpression level of FCRL2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 1.0 times the reference level of FCRL2, theexpression level of NUCKS1 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 0.9 times the reference level of NUCKS1, theexpression level of SHC2 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 0.9 times the reference level of SHC2, theexpression level of SCD5 on CD8⁺ T cells in the PBMCs prior to PD-1blockade is more than 0.9 times the reference level of SCD5, thefrequency of NY-ESO-1^(|)CD8^(|) cells in CD8⁺ T cells in the PBMCsprior to PD-1 blockade treatment is more than 0.5%, the frequency ofMART-1⁺CD8⁺ cells in CD8⁺ T cells in the PBMCs prior to PD-1 blockadetreatment is more than 0.5%, or a combination.
 17. The method of claim2, wherein the PBMCs are also assayed following PD-1 blockade treatmentof the subject, wherein the expression following the PD-1 blockadetreatment is compared to the expression prior to the PD-1 blockadetreatment, wherein an increase in the expression level of LASS6 on CD8⁺T cells in the PBMCs following the PD-1 blockade treatment is positivelyassociated with the expected clinical benefit of the PD-1 blockadetreatment of the subject, wherein a decrease in the expression level ofGADD45B on CD8⁺ T cells in the PBMCs following the PD-1 blockadetreatment is positively associated with the expected clinical benefit ofthe PD-1 blockade treatment of the subject, wherein a decrease in theexpression level of EGR2 on CD8⁺ T cells in the PBMCs following the PD-1blockade treatment is positively associated with the expected clinicalbenefit of the PD-1 blockade treatment of the subject, wherein adecrease in the expression level of EGR1 on CD8⁺ T cells in the PBMCsfollowing the PD-1 blockade treatment is positively associated with theexpected clinical benefit of the PD-1 blockade treatment of the subject,wherein a decrease in the expression level of GADD45G on CD8⁺ T cells inthe PBMCs following the PD-1 blockade treatment is positively associatedwith the expected clinical benefit of the PD-1 blockade treatment of thesubject, wherein a decrease in the expression level of CKS2 on CD8⁺ Tcells in the PBMCs following the PD-1 blockade treatment is positivelyassociated with the expected clinical benefit of the PD-1 blockadetreatment of the subject, wherein a decrease in the expression level ofCDKN1C on CD8⁺ T cells in the PBMCs following the PD-1 blockadetreatment is positively associated with the expected clinical benefit ofthe PD-1 blockade treatment of the subject, wherein a decrease in theexpression level of IER5 on CD8⁺ T cells in the PBMCs following the PD-1blockade treatment is positively associated with the expected clinicalbenefit of the PD-1 blockade treatment of the subject, wherein adecrease in the expression level of ING1 on CD8⁺ T cells in the PBMCsfollowing the PD-1 blockade treatment is positively associated with theexpected clinical benefit of the PD-1 blockade treatment of the subject,wherein a decrease in the expression level of IFIH1 on CD8⁺ T cells inthe PBMCs following the PD-1 blockade treatment is positively associatedwith the expected clinical benefit of the PD-1 blockade treatment of thesubject, wherein a decrease in the expression level of IFNG on CD8⁺ Tcells in the PBMCs following the PD-1 blockade treatment is positivelyassociated with the expected clinical benefit of the PD-1 blockadetreatment of the subject, wherein a decrease in the expression level ofIER5L on CD8⁺ T cells in the PBMCs following the PD-1 blockade treatmentis positively associated with the expected clinical benefit of the PD-1blockade treatment of the subject, wherein an increase in the expressionlevel of NY-ESO-1 on CD8⁺ T cells in the PBMCs following PD-1 blockadetreatment is positively associated with the expected clinical benefit ofPD-1 blockade treatment of the subject, wherein an increase in theexpression level of MART-1 on CD8⁺ T cells in the PBMCs following PD-1blockade treatment is positively associated with the expected clinicalbenefit of PD-1 blockade treatment of the subject, wherein a decrease inthe number or frequency of Tregs cells in CD4⁺ T cells in PBMCsfollowing PD-1 blockade treatment is positively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject,wherein an increase in the number or frequency of Tregs cells in CD4⁺ Tcells in PBMCs following PD-1 blockade treatment is negativelyassociated with the expected clinical benefit of PD-1 blockade treatmentof the subject, wherein a decrease in the number or frequency ofCTLA-4⁺CD4⁺ cells in CD4⁺ T cells in PBMCs following PD-1 blockadetreatment is positively associated with the expected clinical benefit ofPD-1 blockade treatment of the subject, wherein an increase in thenumber or frequency of CTLA-4⁺CD4⁺ cells in CD4⁺ T cells in PBMCsfollowing PD-1 blockade treatment is negatively associated with theexpected clinical benefit of PD-1 blockade treatment of the subject,wherein a decrease in the number or frequency of CTLA-4⁺CD8⁺ cells inCD8⁺ T cells in PBMCs following PD-1 blockade treatment is positivelyassociated with the expected clinical benefit of PD-1 blockade treatmentof the subject, and wherein an increase in the number or frequency ofCTLA-4⁺CD8⁺ cells in CD8⁺ T cells in PBMCs following PD-1 blockadetreatment is negatively associated with the expected clinical benefit ofPD-1 blockade treatment of the subject.
 18. The method of claim 17,wherein an expression level of LASS6 on CD8⁺ T cells in the PBMCsfollowing the PD-1 blockade treatment more than 1.2 times the expressionlevel prior to the PD-1 blockade treatment indicates that the subjectcan expect a clinical benefit from the PD-1 blockade treatment, whereinan expression level of GADD45B on CD8⁺ T cells in the PBMCs followingthe PD-1 blockade treatment less than 0.8 times the expression levelprior to the PD-1 blockade treatment indicates that the subject canexpect a clinical benefit from the PD-1 blockade treatment, wherein anexpression level of EGR2 on CD8⁺ T cells in the PBMCs following the PD-1blockade treatment less than 0.8 times the expression level prior to thePD-1 blockade treatment indicates that the subject can expect a clinicalbenefit from the PD-1 blockade treatment, wherein an expression level ofEGR1 on CD8^(|) T cells in the PBMCs following the PD-1 blockadetreatment less than 0.7 times the expression level prior to the PD-1blockade treatment indicates that the subject can expect a clinicalbenefit from the PD-1 blockade treatment, wherein an expression level ofGADD45G on CD8⁺ T cells in the PBMCs following the PD-1 blockadetreatment less than 0.9 times the expression level prior to the PD-1blockade treatment indicates that the subject can expect a clinicalbenefit from the PD-1 blockade treatment, wherein an expression level ofCKS2 on CD8⁺ T cells in the PBMCs following the PD-1 blockade treatmentless than 0.9 times the expression level prior to the PD-1 blockadetreatment indicates that the subject can expect a clinical benefit fromthe PD-1 blockade treatment, wherein an expression level of CDKN1C onCD8⁺ T cells in the PBMCs following the PD-1 blockade treatment lessthan 0.9 times the expression level prior to the PD-1 blockade treatmentindicates that the subject can expect a clinical benefit from the PD-1blockade treatment, wherein an expression level of IER5 on CD8^(|) Tcells in the PBMCs following the PD-1 blockade treatment less than 0.9times the expression level prior to the PD-1 blockade treatmentindicates that the subject can expect a clinical benefit from the PD-1blockade treatment, wherein an expression level of ING1 on CD8⁺ T cellsin the PBMCs following the PD-1 blockade treatment less than 0.9 timesthe expression level prior to the PD-1 blockade treatment indicates thatthe subject can expect a clinical benefit from the PD-1 blockadetreatment, wherein an expression level of IFIH1 on CD8⁺ T cells in thePBMCs following the PD-1 blockade treatment less than 0.9 times theexpression level prior to the PD-1 blockade treatment indicates that thesubject can expect a clinical benefit from the PD-1 blockade treatment,wherein an expression level of IFNG on CD8⁺ T cells in the PBMCsfollowing the PD-1 blockade treatment less than 0.9 times the expressionlevel prior to the PD-1 blockade treatment indicates that the subjectcan expect a clinical benefit from the PD-1 blockade treatment, andwherein an expression level of IER5L on CD8^(|) T cells in the PBMCsfollowing the PD-1 blockade treatment less than 0.8 times the expressionlevel prior to the PD-1 blockade treatment indicates that the subjectcan expect a clinical benefit from the PD-1 blockade treatment, whereinan expression level of NY-ESO-1 on CD8⁺ T cells in the PBMCs followingPD-1 blockade treatment more than 1.2 times the expression level priorto the PD-1 blockade treatment indicates that the subject can expect aclinical benefit from the PD-1 blockade treatment, wherein an expressionlevel of MART-1 on CD8⁺ T cells in the PBMCs following PD-1 blockadetreatment more than 1.2 times the expression level prior to the PD-1blockade treatment indicates that the subject can expect a clinicalbenefit from the PD-1 blockade treatment, wherein the number orfrequency of Tregs cells in CD4⁺ T cells in PBMCs following the PD-1blockade treatment is less than 1.0 times the number or frequency ofTregs cells in CD4⁺ T cells in PBMCs prior to the PD-1 blockadetreatment indicates that the subject can expect a clinical benefit fromthe PD-1 blockade treatment, wherein the number or frequency ofCTLA-4⁺CD4⁺ cells in CD4⁺ T cells in PBMCs following the PD-1 blockadetreatment is less than 1.0 times the number or frequency of CTLA-4⁺CD4⁺cells in CD4⁺ T cells in PBMCs prior to the PD-1 blockade treatmentindicates that the subject can expect a clinical benefit from the PD-1blockade treatment, and wherein the number or frequency of CTLA-4⁺CD8⁺cells in CD8⁺ T cells in PBMCs following the PD-1 blockade treatment isless than 1.0 times the number or frequency of CTLA-4⁺CD8⁺ cells in CD8⁺T cells in PBMCs prior to the PD-1 blockade treatment indicates that thesubject can expect a clinical benefit from the PD-1 blockade treatment.19. The method of claim 17 or 18 further comprising continuing treatmentof the subject with a PD-1 blockade treatment if the expression level ofLASS6 on CD8⁺ T cells in the PBMCs following the PD-1 blockade treatmentis more than 1.2 times the expression level prior to the PD-1 blockadetreatment, the expression level of GADD45B on CD8⁺ T cells in the PBMCsfollowing the PD-1 blockade treatment is less than 0.8 times theexpression level prior to the PD-1 blockade treatment, the expressionlevel of EGR2 on CD8⁺ T cells in the PBMCs following the PD-1 blockadetreatment is less than 0.8 times the expression level prior to the PD-1blockade treatment, the expression level of EGR1 on CD8⁺ T cells in thePBMCs following the PD-1 blockade treatment is less than 0.7 times theexpression level prior to the PD-1 blockade treatment, the expressionlevel of GADD45G on CD8^(|) T cells in the PBMCs following the PD-1blockade treatment is less than 0.9 times the expression level prior tothe PD-1 blockade treatment, the expression level of CKS2 on CD8⁺ Tcells in the PBMCs following the PD-1 blockade treatment is less than0.9 times the expression level prior to the PD-1 blockade treatment, theexpression level of CDKN1C on CD8⁺ T cells in the PBMCs following thePD-1 blockade treatment is less than 0.9 times the expression levelprior to the PD-1 blockade treatment, the expression level of IER5 onCD8⁺ T cells in the PBMCs following the PD-1 blockade treatment is lessthan 0.9 times the expression level prior to the PD-1 blockadetreatment, the expression level of NY-ESO-1 on CD8⁺ T cells in the PBMCsfollowing PD-1 blockade treatment is more than 1.2 times the expressionlevel prior to the PD-1 blockade treatment, the expression level ofMART-1 on CD8⁺ T cells in the PBMCs following PD-1 blockade treatment ismore than 1.2 times the expression level prior to the PD-1 blockadetreatment, the number or frequency of Tregs cells in CD4⁺ T cells inPBMCs following the PD-1 blockade treatment is less than 1.0 times thenumber or frequency of Tregs cells in CD4⁺ T cells in PBMCs prior to thePD-1 blockade treatment, the number or frequency of CTLA-4⁺CD4^(|) cellsin CD4⁺ T cells in PBMCs following the PD-1 blockade treatment is lessthan 1.0 times the number or frequency of CTLA-4⁺CD4⁺ cells in CD4⁺ Tcells in PBMCs prior to the PD-1 blockade treatment, the number orfrequency of CTLA-4⁺CD8⁺ cells in CD8⁺ T cells in PBMCs following thePD-1 blockade treatment is less than 1.0 times the number or frequencyof CTLA-4⁺CD8⁺ cells in CD8⁺ T cells in PBMCs prior to the PD-1 blockadetreatment, or a combination.
 20. The method of claim 17 or 18 furthercomprising treating the subject with a treatment other than a PD-1blockade treatment if the expression level of LASS6 on CD8⁺ T cells inthe PBMCs following the PD-1 blockade treatment is less than 1.1 timesthe expression level prior to the PD-1 blockade treatment, theexpression level of GADD45B on CD8⁺ T cells in the PBMCs following thePD-1 blockade treatment is more than 0.9 times the expression levelprior to the PD-1 blockade treatment, the expression level of EGR2 onCD8⁺ T cells in the PBMCs following the PD-1 blockade treatment is morethan 0.9 times the expression level prior to the PD-1 blockadetreatment, the expression level of EGR1 on CD8⁺ T cells in the PBMCsfollowing the PD-1 blockade treatment is more than 0.8 times theexpression level prior to the PD-1 blockade treatment, the expressionlevel of GADD45G on CD8⁺ T cells in the PBMCs following the PD-1blockade treatment is more than 1.0 times the expression level prior tothe PD-1 blockade treatment, the expression level of CKS2 on CD8⁺ Tcells in the PBMCs following the PD-1 blockade treatment is more than1.0 times the expression level prior to the PD-1 blockade treatment, theexpression level of CDKN1C on CD8⁺ T cells in the PBMCs following thePD-1 blockade treatment is more than 1.0 times the expression levelprior to the PD-1 blockade treatment, the expression level of IER5 onCD8⁺ T cells in the PBMCs following the PD-1 blockade treatment is morethan 1.0 times the expression level prior to the PD-1 blockadetreatment, the expression level of NY-ESO-1 on CD8⁺ T cells in the PBMCsfollowing PD-1 blockade treatment is less than 1.0 times the expressionlevel prior to the PD-1 blockade treatment, the expression level ofMART-1 on CD8⁺ T cells in the PBMCs following PD-1 blockade treatment isless than 1.0 times the expression level prior to the PD-1 blockadetreatment, the number or frequency of Tregs cells in CD4⁺ T cells inPBMCs following PD-1 blockade treatment is more than 1.0 times thenumber or frequency of Tregs cells in CD4⁺ T cells in PBMCs prior to thePD-1 blockade treatment, the number or frequency of CTLA-4⁺CD4⁺ cells inCD4⁺ T cells in PBMCs following the PD-1 blockade treatment is more than1.0 times the number or frequency of CTLA-4⁺CD4⁺ cells in CD4⁺ T cellsin PBMCs prior to the PD-1 blockade treatment, the number or frequencyof CTLA-4⁺CD8⁺ cells in CD8⁺ T cells in PBMCs following the PD-1blockade treatment is more than 1.0 times the number or frequency ofCTLA-4⁺CD8⁺ cells in CD8⁺ T cells in PBMCs prior to the PD-1 blockadetreatment, or a combination.
 21. The method of claim 17 or 18 furthercomprising treating the subject with both a PD-1 blockade treatment anda different treatment if the expression level of LASS6 on CD8⁺ T cellsin the PBMCs following the PD-1 blockade treatment is less than 1.1times the expression level prior to the PD-1 blockade treatment, theexpression level of GADD45B on CD8⁺ T cells in the PBMCs following thePD-1 blockade treatment is more than 0.9 times the expression levelprior to the PD-1 blockade treatment, the expression level of EGR2 onCD8⁺ T cells in the PBMCs following the PD-1 blockade treatment is morethan 0.9 times the expression level prior to the PD-1 blockadetreatment, the expression level of EGR1 on CD8⁺ T cells in the PBMCsfollowing the PD-1 blockade treatment is more than 0.8 times theexpression level prior to the PD-1 blockade treatment, the expressionlevel of GADD45G on CD8⁺ T cells in the PBMCs following the PD-1blockade treatment is more than 1.0 times the expression level prior tothe PD-1 blockade treatment, the expression level of CKS2 on CD8^(|) Tcells in the PBMCs following the PD-1 blockade treatment is more than1.0 times the expression level prior to the PD-1 blockade treatment, theexpression level of CDKN1C on CD8⁺ T cells in the PBMCs following thePD-1 blockade treatment is more than 1.0 times the expression levelprior to the PD-1 blockade treatment, the expression level of IER5 onCD8⁺ T cells in the PBMCs following the PD-1 blockade treatment is morethan 1.0 times the expression level prior to the PD-1 blockadetreatment, the expression level of NY-ESO-1 on CD8⁺ T cells in the PBMCsfollowing PD-1 blockade treatment is less than 1.0 times the expressionlevel prior to the PD-1 blockade treatment, the expression level ofMART-1 on CD8⁺ T cells in the PBMCs following PD-1 blockade treatment isless than 1.0 times the expression level prior to the PD-1 blockadetreatment, the number or frequency of Tregs cells in CD4⁺ T cells inPBMCs following PD-1 blockade treatment is more than 1.0 times thenumber or frequency of Tregs cells in CD4⁺ T cells in PBMCs prior to thePD-1 blockade treatment, the number or frequency of CTLA-4⁺CD4⁺ cells inCD4⁺ T cells in PBMCs following the PD-1 blockade treatment is more than1.0 times the number or frequency of CTLA-4⁺CD4⁺ cells in CD4⁺ T cellsin PBMCs prior to the PD-1 blockade treatment, the number or frequencyof CTLA-4^(|)CD8^(|) cells in CD8^(|) T cells in PBMCs following thePD-1 blockade treatment is more than 1.0 times the number or frequencyof CTLA-4⁺CD8⁺ cells in CD8⁺ T cells in PBMCs prior to the PD-1 blockadetreatment, or a combination.
 22. The method of claim 17 or 18 furthercomprising selecting a subject for continued PD-1 blockade treatment ifthe expression level of LASS6 on CD8⁺ T cells in the PBMCs following thePD-1 blockade treatment is more than 1.2 times the expression levelprior to the PD-1 blockade treatment, the expression level of GADD45B onCD8⁺ T cells in the PBMCs following the PD-1 blockade treatment is lessthan 0.8 times the expression level prior to the PD-1 blockadetreatment, the expression level of EGR2 on CD8⁺ T cells in the PBMCsfollowing the PD-1 blockade treatment is less than 0.8 times theexpression level prior to the PD-1 blockade treatment, the expressionlevel of EGR1 on CD8⁺ T cells in the PBMCs following the PD-1 blockadetreatment is less than 0.7 times the expression level prior to the PD-1blockade treatment, the expression level of GADD45G on CD8^(|) T cellsin the PBMCs following the PD-1 blockade treatment is less than 0.9times the expression level prior to the PD-1 blockade treatment, theexpression level of CKS2 on CD8⁺ T cells in the PBMCs following the PD-1blockade treatment is less than 0.9 times the expression level prior tothe PD-1 blockade treatment, the expression level of CDKN1C on CD8⁺ Tcells in the PBMCs following the PD-1 blockade treatment is less than0.9 times the expression level prior to the PD-1 blockade treatment, theexpression level of IER5 on CD8⁺ T cells in the PBMCs following the PD-1blockade treatment is less than 0.9 times the expression level prior tothe PD-1 blockade treatment, the expression level of NY-ESO-1 on CD8⁺ Tcells in the PBMCs following PD-1 blockade treatment is more than 1.2times the expression level prior to the PD-1 blockade treatment, theexpression level of MART-1 on CD8⁺ T cells in the PBMCs following PD-1blockade treatment is more than 1.2 times the expression level prior tothe PD-1 blockade treatment, the number or frequency of Tregs cells inCD4⁺ T cells in PBMCs following the PD-1 blockade treatment is less than1.0 times the number or frequency of Tregs cells in CD4⁺ T cells inPBMCs prior to the PD-1 blockade treatment, the number or frequency ofCTLA-4⁺CD4⁺ cells in CD4⁺ T cells in PBMCs following the PD-1 blockadetreatment is less than 1.0 times the number or frequency of CTLA-4⁺CD4⁺cells in CD4⁺ T cells in PBMCs prior to the PD-1 blockade treatment, thenumber or frequency of CTLA-4⁺CD8⁺ cells in CD8⁺ T cells in PBMCsfollowing the PD-1 blockade treatment is less than 1.0 times the numberor frequency of CTLA-4⁺CD8⁺ cells in CD8⁺ T cells in PBMCs prior to thePD-1 blockade treatment, or a combination.
 23. The method of claim 17 or18 further comprising selecting a subject for a treatment other than aPD-1 blockade treatment if the expression level of LASS6 on CD8⁺ T cellsin the PBMCs following the PD-1 blockade treatment is less than 1.1times the expression level prior to the PD-1 blockade treatment, theexpression level of GADD45B on CD8⁺ T cells in the PBMCs following thePD-1 blockade treatment is more than 0.9 times the expression levelprior to the PD-1 blockade treatment, the expression level of EGR2 onCD8⁺ T cells in the PBMCs following the PD-1 blockade treatment is morethan 0.9 times the expression level prior to the PD-1 blockadetreatment, the expression level of EGR1 on CD8⁺ T cells in the PBMCsfollowing the PD-1 blockade treatment is more than 0.8 times theexpression level prior to the PD-1 blockade treatment, the expressionlevel of GADD45G on CD8⁺ T cells in the PBMCs following the PD-1blockade treatment is more than 1.0 times the expression level prior tothe PD-1 blockade treatment, the expression level of CKS2 on CD8⁺ Tcells in the PBMCs following the PD-1 blockade treatment is more than1.0 times the expression level prior to the PD-1 blockade treatment, theexpression level of CDKN1C on CD8⁺ T cells in the PBMCs following thePD-1 blockade treatment is more than 1.0 times the expression levelprior to the PD-1 blockade treatment, the expression level of IER5 onCD8⁺ T cells in the PBMCs following the PD-1 blockade treatment is morethan 1.0 times the expression level prior to the PD-1 blockadetreatment, the expression level of NY-ESO-1 on CD8⁺ T cells in the PBMCsfollowing PD-1 blockade treatment is less than 1.0 times the expressionlevel prior to the PD-1 blockade treatment, the expression level ofMART-1 on CD8⁺ T cells in the PBMCs following PD-1 blockade treatment isless than 1.0 times the expression level prior to the PD-1 blockadetreatment, the number or frequency of Tregs cells in CD4⁺ T cells inPBMCs following PD-1 blockade treatment is more than 1.0 times thenumber or frequency of Tregs cells in CD4⁺ T cells in PBMCs prior to thePD-1 blockade treatment, the number or frequency of CTLA-4⁺CD4⁺ cells inCD4⁺ T cells in PBMCs following the PD-1 blockade treatment is more than1.0 times the number or frequency of CTLA-4⁺CD4⁺ cells in CD4⁺ T cellsin PBMCs prior to the PD-1 blockade treatment, the number or frequencyof CTLA-4⁺CD8⁺ cells in CD8⁺ T cells in PBMCs following the PD-1blockade treatment is more than 1.0 times the number or frequency ofCTLA-4^(|)CD8^(|) cells in CD8^(|) T cells in PBMCs prior to the PD-1blockade treatment, or a combination.
 24. The method of claim 17 or 18further comprising selecting a subject for treatment with both a PD-1blockade treatment and a different treatment if the expression level ofLASS6 on CD8⁺ T cells in the PBMCs following the PD-1 blockade treatmentis less than 1.1 times the expression level prior to the PD-1 blockadetreatment, the expression level of GADD45B on CD8⁺ T cells in the PBMCsfollowing the PD-1 blockade treatment is more than 0.9 times theexpression level prior to the PD-1 blockade treatment, the expressionlevel of EGR2 on CD8⁺ T cells in the PBMCs following the PD-1 blockadetreatment is more than 0.9 times the expression level prior to the PD-1blockade treatment, the expression level of EGR1 on CD8⁺ T cells in thePBMCs following the PD-1 blockade treatment is more than 0.8 times theexpression level prior to the PD-1 blockade treatment, the expressionlevel of GADD45G on CD8⁺ T cells in the PBMCs following the PD-1blockade treatment is more than 1.0 times the expression level prior tothe PD-1 blockade treatment, the expression level of CKS2 on CD8⁺ Tcells in the PBMCs following the PD-1 blockade treatment is more than1.0 times the expression level prior to the PD-1 blockade treatment, theexpression level of CDKN1C on CD8⁺ T cells in the PBMCs following thePD-1 blockade treatment is more than 1.0 times the expression levelprior to the PD-1 blockade treatment, the expression level of IER5 onCD8⁺ T cells in the PBMCs following the PD-1 blockade treatment is morethan 1.0 times the expression level prior to the PD-1 blockadetreatment, the expression level of NY-ESO-1 on CD8⁺ T cells in the PBMCsfollowing PD-1 blockade treatment is less than 1.0 times the expressionlevel prior to the PD-1 blockade treatment, the expression level ofMART-1 on CD8⁺ T cells in the PBMCs following PD-1 blockade treatment isless than 1.0 times the expression level prior to the PD-1 blockadetreatment, the number or frequency of Tregs cells in CD4⁺ T cells inPBMCs following PD-1 blockade treatment is more than 1.0 times thenumber or frequency of Tregs cells in CD4⁺ T cells in PBMCs prior to thePD-1 blockade treatment, the number or frequency of CTLA-4⁺CD4⁺ cells inCD4⁺ T cells in PBMCs following the PD-1 blockade treatment is more than1.0 times the number or frequency of CTLA-4⁺CD4⁺ cells in CD4⁺ T cellsin PBMCs prior to the PD-1 blockade treatment, the number or frequencyof CTLA-4⁺CD8⁺ cells in CD8⁺ T cells in PBMCs following the PD-1blockade treatment is more than 1.0 times the number or frequency ofCTLA-4⁺CD8⁺ cells in CD8⁺ T cells in PBMCs prior to the PD-1 blockadetreatment, or a combination.
 25. The method of claim 17 or 18, whereinthe subject is further treated with a PD-1 blockade treatment if theexpression level of LASS6 on CD8⁺ T cells in the PBMCs following thePD-1 blockade treatment is more than 1.2 times the expression levelprior to the PD-1 blockade treatment, the expression level of GADD45B onCD8⁺ T cells in the PBMCs following the PD-1 blockade treatment is lessthan 0.8 times the expression level prior to the PD-1 blockadetreatment, the expression level of EGR2 on CD8⁺ T cells in the PBMCsfollowing the PD-1 blockade treatment is less than 0.8 times theexpression level prior to the PD-1 blockade treatment, the expressionlevel of EGR1 on CD8⁺ T cells in the PBMCs following the PD-1 blockadetreatment is less than 0.7 times the expression level prior to the PD-1blockade treatment, the expression level of GADD45G on CD8⁺ T cells inthe PBMCs following the PD-1 blockade treatment is less than 0.9 timesthe expression level prior to the PD-1 blockade treatment, theexpression level of CKS2 on CD8⁺ T cells in the PBMCs following the PD-1blockade treatment is less than 0.9 times the expression level prior tothe PD-1 blockade treatment, the expression level of CDKN1C on CD8⁺ Tcells in the PBMCs following the PD-1 blockade treatment is less than0.9 times the expression level prior to the PD-1 blockade treatment, theexpression level of IER5 on CD8^(|) T cells in the PBMCs following thePD-1 blockade treatment is less than 0.9 times the expression levelprior to the PD-1 blockade treatment, the expression level of NY-ESO-1on CD8⁺ T cells in the PBMCs following PD-1 blockade treatment is morethan 1.2 times the expression level prior to the PD-1 blockadetreatment, the expression level of MART-1 on CD8⁺ T cells in the PBMCsfollowing PD-1 blockade treatment is more than 1.2 times the expressionlevel prior to the PD-1 blockade treatment, the number or frequency ofTregs cells in CD4⁺ T cells in PBMCs following the PD-1 blockadetreatment is less than 1.0 times the number or frequency of Tregs cellsin CD4⁺ T cells in PBMCs prior to the PD-1 blockade treatment, thenumber or frequency of CTLA-4⁺CD4⁺ cells in CD4⁺ T cells in PBMCsfollowing the PD-1 blockade treatment is less than 1.0 times the numberor frequency of CTLA-4⁺CD4⁺ cells in CD4⁺ T cells in PBMCs prior to thePD-1 blockade treatment, the number or frequency of CTLA-4⁺CD8⁺ cells inCD8⁺ T cells in PBMCs following the PD-1 blockade treatment is less than1.0 times the number or frequency of CTLA-4⁺CD8⁺ cells in CD8⁺ T cellsin PBMCs prior to the PD-1 blockade treatment, or a combination.
 26. Themethod of claim 17 or 18, wherein the subject is not further treatedwith a PD-1 blockade treatment if the expression level of LASS6 on CD8⁺T cells in the PBMCs following the PD-1 blockade treatment is less than1.1 times the expression level prior to the PD-1 blockade treatment, theexpression level of GADD45B on CD8⁺ T cells in the PBMCs following thePD-1 blockade treatment is more than 0.9 times the expression levelprior to the PD-1 blockade treatment, the expression level of EGR2 onCD8⁺ T cells in the PBMCs following the PD-1 blockade treatment is morethan 0.9 times the expression level prior to the PD-1 blockadetreatment, the expression level of EGR1 on CD8⁺ T cells in the PBMCsfollowing the PD-1 blockade treatment is more than 0.8 times theexpression level prior to the PD-1 blockade treatment, the expressionlevel of GADD45G on CD8⁺ T cells in the PBMCs following the PD-1blockade treatment is more than 1.0 times the expression level prior tothe PD-1 blockade treatment, the expression level of CKS2 on CD8⁺ Tcells in the PBMCs following the PD-1 blockade treatment is more than1.0 times the expression level prior to the PD-1 blockade treatment, theexpression level of CDKN1C on CD8^(|) T cells in the PBMCs following thePD-1 blockade treatment is more than 1.0 times the expression levelprior to the PD-1 blockade treatment, the expression level of IER5 onCD8⁺ T cells in the PBMCs following the PD-1 blockade treatment is morethan 1.0 times the expression level prior to the PD-1 blockadetreatment, the expression level of NY-ESO-1 on CD8⁺ T cells in the PBMCsfollowing PD-1 blockade treatment is less than 1.0 times the expressionlevel prior to the PD-1 blockade treatment, the expression level ofMART-1 on CD8⁺ T cells in the PBMCs following PD-1 blockade treatment isless than 1.0 times the expression level prior to the PD-1 blockadetreatment, the number or frequency of Tregs cells in CD4⁺ T cells inPBMCs following PD-1 blockade treatment is more than 1.0 times thenumber or frequency of Tregs cells in CD4⁺ T cells in PBMCs prior to thePD-1 blockade treatment, the number or frequency of CTLA-4⁺CD4⁺ cells inCD4⁺ T cells in PBMCs following the PD-1 blockade treatment is more than1.0 times the number or frequency of CTLA-4⁺CD4⁺ cells in CD4⁺ T cellsin PBMCs prior to the PD-1 blockade treatment, the number or frequencyof CTLA-4⁺CD8⁺ cells in CD8⁺ T cells in PBMCs following the PD-1blockade treatment is more than 1.0 times the number or frequency ofCTLA-4⁺CD8⁺ cells in CD8⁺ T cells in PBMCs prior to the PD-1 blockadetreatment, or a combination.
 27. A method of treating a subjectdiagnosed with melanoma with a PD-1 blockade treatment if the expressionlevel of LASS6 on CD8⁺ T cells in the PBMCs following the PD-1 blockadetreatment is more than 1.2 times the expression level prior to the PD-1blockade treatment, the expression level of GADD45B on CD8⁺ T cells inthe PBMCs following the PD-1 blockade treatment is less than 0.8 timesthe expression level prior to the PD-1 blockade treatment, theexpression level of EGR2 on CD8⁺ T cells in the PBMCs following the PD-1blockade treatment is less than 0.8 times the expression level prior tothe PD-1 blockade treatment, the expression level of EGR1 on CD8⁺ Tcells in the PBMCs following the PD-1 blockade treatment is less than0.7 times the expression level prior to the PD-1 blockade treatment, theexpression level of GADD45G on CD8⁺ T cells in the PBMCs following thePD-1 blockade treatment is less than 0.9 times the expression levelprior to the PD-1 blockade treatment, the expression level of CKS2 onCD8⁺ T cells in the PBMCs following the PD-1 blockade treatment is lessthan 0.9 times the expression level prior to the PD-1 blockadetreatment, the expression level of CDKN1C on CD8⁺ T cells in the PBMCsfollowing the PD-1 blockade treatment is less than 0.9 times theexpression level prior to the PD-1 blockade treatment, the expressionlevel of IER5 on CD8⁺ T cells in the PBMCs following the PD-1 blockadetreatment is less than 0.9 times the expression level prior to the PD-1blockade treatment, the expression level of NY-ESO-1 on CD8^(|) T cellsin the PBMCs following PD-1 blockade treatment is more than 1.2 timesthe expression level prior to the PD-1 blockade treatment, theexpression level of MART-1 on CD8⁺ T cells in the PBMCs following PD-1blockade treatment is more than 1.2 times the expression level prior tothe PD-1 blockade treatment, the number or frequency of Tregs cells inCD4⁺ T cells in PBMCs following the PD-1 blockade treatment is less than1.0 times the number or frequency of Tregs cells in CD4⁺ T cells inPBMCs prior to the PD-1 blockade treatment, the number or frequency ofCTLA-4^(|)CD4^(|) cells in CD4^(|) T cells in PBMCs following the PD-1blockade treatment is less than 1.0 times the number or frequency ofCTLA-4⁺CD4⁺ cells in CD4⁺ T cells in PBMCs prior to the PD-1 blockadetreatment, the number or frequency of CTLA-4⁺CD8⁺ cells in CD8⁺ T cellsin PBMCs following the PD-1 blockade treatment is less than 1.0 timesthe number or frequency of CTLA-4⁺CD8⁺ cells in CD8⁺ T cells in PBMCsprior to the PD-1 blockade treatment, or a combination.
 28. A method oftreating a subject diagnosed with melanoma with a treatment other than aPD-1 blockade treatment if the expression level of LASS6 on CD8^(|) Tcells in the PBMCs following the PD-1 blockade treatment is less than1.1 times the expression level prior to the PD-1 blockade treatment, theexpression level of GADD45B on CD8⁺ T cells in the PBMCs following thePD-1 blockade treatment is more than 0.9 times the expression levelprior to the PD-1 blockade treatment, the expression level of EGR2 onCD8⁺ T cells in the PBMCs following the PD-1 blockade treatment is morethan 0.9 times the expression level prior to the PD-1 blockadetreatment, the expression level of EGR1 on CD8⁺ T cells in the PBMCsfollowing the PD-1 blockade treatment is more than 0.8 times theexpression level prior to the PD-1 blockade treatment, the expressionlevel of GADD45G on CD8⁺ T cells in the PBMCs following the PD-1blockade treatment is more than 1.0 times the expression level prior tothe PD-1 blockade treatment, the expression level of CKS2 on CD8⁺ Tcells in the PBMCs following the PD-1 blockade treatment is more than1.0 times the expression level prior to the PD-1 blockade treatment, theexpression level of CDKN1C on CD8⁺ T cells in the PBMCs following thePD-1 blockade treatment is more than 1.0 times the expression levelprior to the PD-1 blockade treatment, the expression level of IER5 onCD8⁺ T cells in the PBMCs following the PD-1 blockade treatment is morethan 1.0 times the expression level prior to the PD-1 blockadetreatment, the expression level of NY-ESO-1 on CD8⁺ T cells in the PBMCsfollowing PD-1 blockade treatment is less than 1.0 times the expressionlevel prior to the PD-1 blockade treatment, the expression level ofMART-1 on CD8⁺ T cells in the PBMCs following PD-1 blockade treatment isless than 1.0 times the expression level prior to the PD-1 blockadetreatment, the number or frequency of Tregs cells in CD4⁺ T cells inPBMCs following PD-1 blockade treatment is more than 1.0 times thenumber or frequency of Tregs cells in CD4^(|) T cells in PBMCs prior tothe PD-1 blockade treatment, the number or frequency of CTLA-4⁺CD4⁺cells in CD4⁺ T cells in PBMCs following the PD-1 blockade treatment ismore than 1.0 times the number or frequency of CTLA-4⁺CD4⁺ cells in CD4⁺T cells in PBMCs prior to the PD-1 blockade treatment, the number orfrequency of CTLA-4⁺CD8⁺ cells in CD8⁺ T cells in PBMCs following thePD-1 blockade treatment is more than 1.0 times the number or frequencyof CTLA-4⁺CD8⁺ cells in CD8⁺ T cells in PBMCs prior to the PD-1 blockadetreatment, or a combination.
 29. A method of treating a subjectdiagnosed with melanoma with both a PD-1 blockade treatment and adifferent treatment if the expression level of LASS6 on CD8⁺ T cells inthe PBMCs following the PD-1 blockade treatment is less than 1.1 timesthe expression level prior to the PD-1 blockade treatment, theexpression level of GADD45B on CD8⁺ T cells in the PBMCs following thePD-1 blockade treatment is more than 0.9 times the expression levelprior to the PD-1 blockade treatment, the expression level of EGR2 onCD8⁺ T cells in the PBMCs following the PD-1 blockade treatment is morethan 0.9 times the expression level prior to the PD-1 blockadetreatment, the expression level of EGR1 on CD8⁺ T cells in the PBMCsfollowing the PD-1 blockade treatment is more than 0.8 times theexpression level prior to the PD-1 blockade treatment, the expressionlevel of GADD45G on CD8⁺ T cells in the PBMCs following the PD-1blockade treatment is more than 1.0 times the expression level prior tothe PD-1 blockade treatment, the expression level of CKS2 on CD8⁺ Tcells in the PBMCs following the PD-1 blockade treatment is more than1.0 times the expression level prior to the PD-1 blockade treatment, theexpression level of CDKN1C on CD8⁺ T cells in the PBMCs following thePD-1 blockade treatment is more than 1.0 times the expression levelprior to the PD-1 blockade treatment, the expression level of IER5 onCD8⁺ T cells in the PBMCs following the PD-1 blockade treatment is morethan 1.0 times the expression level prior to the PD-1 blockadetreatment, the expression level of NY-ESO-1 on CD8⁺ T cells in the PBMCsfollowing PD-1 blockade treatment is less than 1.0 times the expressionlevel prior to the PD-1 blockade treatment, the expression level ofMART-1 on CD8^(|) T cells in the PBMCs following PD-1 blockade treatmentis less than 1.0 times the expression level prior to the PD-1 blockadetreatment, the number or frequency of Tregs cells in CD4⁺ T cells inPBMCs following PD-1 blockade treatment is more than 1.0 times thenumber or frequency of Tregs cells in CD4⁺ T cells in PBMCs prior to thePD-1 blockade treatment, the number or frequency of CTLA-4⁺CD4⁺ cells inCD4⁺ T cells in PBMCs following the PD-1 blockade treatment is more than1.0 times the number or frequency of CTLA-4⁺CD4⁺ cells in CD4⁺ T cellsin PBMCs prior to the PD-1 blockade treatment, the number or frequencyof CTLA-4⁺CD8⁺ cells in CD8⁺ T cells in PBMCs following the PD-1blockade treatment is more than 1.0 times the number or frequency ofCTLA-4⁺CD8⁺ cells in CD8⁺ T cells in PBMCs prior to the PD-1 blockadetreatment, or a combination.
 30. The method of any one of claims 1 to29, wherein the PD-1 blockade treatment is treatment with a PD-1blocking antibody.
 31. The method of any one of claims 1-30, wherein thePD-1 blockade treatment is treatment with PD-1 blocking antibodyBMS-936558 or BMS-936559.
 32. A method comprising assaying peripheralblood mononuclear cells (PBMCs) from a subject diagnosed with melanomafor expression of (a) CD8, Ki67, and eomesodermin (EOMES); (b) CD8 andEOMES; (c) CD8 and transforming growth factor beta receptor ITT(TGFβR3); (d) CD4 and C-C chemokine receptor type 7 (CCR7); (e) CD4,Ki67, and EOMES; (f) CD4 and CD71; (g) CD8 and CD109; or a combination,wherein the frequency of Ki67⁺EOMES⁺CD8⁺ T cells in CD8⁺ T cells in thePBMCs is inversely associated with the risk of relapse after CTLA-4blockade treatment, wherein the frequency of EOMES⁺CD8⁺ T cells in CD8⁺T cells in the PBMCs is inversely associated with the risk of relapseafter CTLA-4 blockade treatment, wherein the expression level of CCR7 onCD4⁺ T cells in the PBMCs is directly associated with the risk ofrelapse after CTLA-4 blockade treatment, wherein the frequency ofKi67⁺EOMES⁺CD4⁺ T cells in CD4⁺ T cells in the PBMCs is inverselyassociated with the risk of an immune related adverse event (irAE) afterCTLA-4 blockade treatment, wherein the expression level of TGFβR3 onCD8^(|) T cells in the PBMCs is directly associated with the risk of anirAE after CTLA-4 blockade treatment, wherein the expression level ofCD71 on CD4⁺ T cells in the PBMCs is inversely associated with the riskof an irAE after CTLA-4 blockade treatment, wherein the frequency ofCD109⁺CD8⁺ T cells in CD8⁺ T cells in the PBMCs is directly associatedwith the risk of relapse after CTLA-4 blockade treatment, wherein thefrequency of CD71⁺CD4⁺ T cells in CD4⁺ T cells in the PBMCs is inverselyassociated with the risk of an irAE after CTLA-4 blockade treatment. 33.The method of claim 32 further comprising treating the subject with aCTLA-4 blockade treatment if (a) the frequency of Ki67⁺EOMES⁺CD8⁺ Tcells is at least 2.2%, (b) the frequency of EOMES^(|)CD8^(|) T cells isat least 56%, (c) the expression level of CCR7 on CD4⁺ T cells is lowerthan 2400, (d) the frequency of Ki67⁺EOMES⁺CD4⁺ T cells is at least0.45%, (e) the expression level of TGFβR3 on CD8⁺ T cells is lower than525, (f) the expression level of CD109 on CD8⁺ T cells is less than0.7316%, (g) the frequency of CD71⁺CD4⁺ T cells in CD4⁺ T cells in thePBMCs is at least 2.80%, or a combination.
 34. The method of claim 32further comprising treating the subject with a treatment other than aCTLA-4 blockade treatment if (a) the frequency of Ki67⁺EOMES⁺CD8⁺ Tcells is less than 2.11%, (b) the frequency of EOMES' CD8⁺ T cells isless than 55.6%, (c) the expression level of CCR7 on CD4⁺ T cells isgreater than 2402, (d) the frequency of Ki67⁺EOMES⁺CD4⁺ T cells is lessthan 0.446%, (e) the expression level of TGFβR3 on CD8⁺ T cells isgreater than 527, (f) the expression level of CD109 on CD8⁺ T cells isgreater than 0.7315%, (g) the frequency of CD71⁺CD4⁺ T cells in CD4⁺ Tcells in the PBMCs is less than 2.80%, or a combination.
 35. The methodof claim 32 further comprising treating the subject with both a CTLA-4blockade treatment and a different treatment if (a) the frequency ofKi67⁺EOMES⁺CD8⁺ T cells is less than 2.11%, (b) the frequency ofEOMES^(|)CD8^(|) T cells is less than 55.6%, (c) the expression level ofCCR7 on CD4⁺ T cells is greater than 2402, (d) the frequency ofKi67⁺EOMES⁺CD4⁺ T cells is less than 0.446%, (e) the expression level ofTGFβR3 on CD8⁺ T cells is greater than 527, (f) the expression level ofCD109 on CD8⁺ T cells is greater than 0.7315%, (g) the frequency ofCD71⁺CD4⁺ T cells in CD4⁺ T cells in the PBMCs is less than 2.80%, or acombination.
 36. The method of claim 32 further comprising selecting asubject for CTLA-4 blockade treatment if (a) the frequency ofKi67⁺EOMES⁺CD8⁺ T cells is at least 2.2%, (b) the frequency ofEOMES⁺CD8⁺ T cells is at least 56%, (c) the expression level of CCR7 onCD4⁺ T cells is lower than 2400, (d) the frequency of Ki67⁺EOMES⁺CD4⁺ Tcells is at least 0.45%, (e) the expression level of TGFβR3 on CD8⁺ Tcells is lower than 525, (f) the expression level of CD109 on CD8⁺ Tcells is less than 0.7316%, (g) the frequency of CD71^(|)CD4^(|) T cellsin CD4⁺ T cells in the PBMCs is at least 2.80%, or a combination. 37.The method of claim 32 further comprising selecting a subject for atreatment other than a CTLA-4 blockade treatment if (a) the frequency ofKi67⁺EOMES⁺CD8⁺ T cells is less than 2.11%, (b) the frequency ofEOMES⁺CD8⁺ T cells is less than 55.6%, (c) the expression level of CCR7on CD4⁺ T cells is greater than 2402, (d) the frequency ofKi67⁺EOMES⁺CD4⁺ T cells is less than 0.446%, (e) the expression level ofTGFβR3 on CD8⁺ T cells is greater than 527, (f) the expression level ofCD109 on CD8⁺ T cells is greater than 0.7315%, (g) the frequency ofCD71^(|)CD4⁺ T cells in CD4⁺ T cells in the PBMCs is less than 2.80%, ora combination.
 38. The method of claim 32 further comprising selecting asubject for treatment with both a CTLA-4 blockade treatment and adifferent treatment if (a) the frequency of Ki67⁺EOMES⁺CD8⁺ T cells isless than 2.11%, (b) the frequency of EOMES⁺CD8⁺ T cells is less than55.6%, (c) the expression level of CCR7 on CD4⁺ T cells is greater than2402, (d) the frequency of Ki67⁺EOMES⁺CD4⁺ T cells is less than 0.446%,(e) the expression level of TGFβR3 on CD8⁺ T cells is greater than 527,(f) the expression level of CD109 on CD8^(|) T cells is greater than0.7315%, (g) the frequency of CD71^(|)CD4^(|) T cells in CD4⁺ T cells inthe PBMCs is less than 2.80%, or a combination.
 39. The method of claim32, wherein the subject is treated with a CTLA-4 blockade treatment if(a) the frequency of Ki67⁺EOMES⁺CD8⁺ T cells is at least 2.2%, (b) thefrequency of EOMES⁺CD8⁺ T cells is at least 56%, (c) the expressionlevel of CCR7 on CD4⁺ T cells is lower than 2400, (d) the frequency ofKi67⁺EOMES⁺CD4⁺ T cells is at least 0.45%, (c) the expression level ofTGFβR3 on CD8⁺ T cells is lower than 525, (f) the expression level ofCD109 on CD8⁺ T cells is less than 0.7316%, (g) the frequency ofCD71⁺CD4⁺ T cells in CD4⁺ T cells in the PBMCs is at least 2.80%, or acombination.
 40. The method of claim 32, wherein the subject is nottreated with a CTLA-4 blockade treatment if (a) the frequency ofKi67⁺EOMES⁺CD8⁺ T cells is less than 2.11%, (b) the frequency ofEOMES⁺CD8⁺ T cells is less than 55.6%, (c) the expression level of CCR7on CD4⁺ T cells is greater than 2402, (d) the frequency ofKi67⁺EOMES⁺CD4⁺ T cells is less than 0.446%, (e) the expression level ofTGFβR3 on CD8⁺ T cells is greater than 527, (f) the expression level ofCD109 on CD8⁺ T cells is greater than 0.7315%, (g) the frequency ofCD71⁺CD4⁺ T cells in CD4⁺ T cells in the PBMCs is less than 2.80%, or acombination.
 41. A method of treating a subject diagnosed with melanomawith a CTLA-4 blockade treatment if (a) the measured frequency ofKi67⁺EOMES⁺CD8⁺ T cells in peripheral blood mononuclear cells (PBMCs) ofthe subject is at least 2.2%, (b) the measured frequency of EOMES⁺CD8⁺ Tcells in PBMCs of the subject is at least 56%, (c) the measuredexpression level of CCR7 on CD4⁺ T cells in PBMCs of the subject islower than 2400, (d) the measured frequency of Ki67⁺EOMES⁺CD4⁺ T cellsin PBMCs of the subject is at least 0.45%, (e) the measured expressionlevel of TGFβR3 on CD8⁺ T cells in PBMCs of the subject is lower than525, (f) the expression level of CD109 on CD8⁺ T cells is less than0.7316%, (g) the frequency of CD71⁺CD4⁺ T cells in CD4⁺ T cells in thePBMCs is at least 2.80%, or a combination.
 42. A method of treating asubject diagnosed with melanoma with a treatment other than a CTLA-4blockade treatment if (a) the measured frequency of Ki67⁺EOMES⁺CD8⁺ Tcells in peripheral blood mononuclear cells (PBMCs) of the subject isless than 2.11%, (b) the measured frequency of EOMES⁺CD8⁺ T cells inPBMCs of the subject is less than 55.6%, (c) the measured expressionlevel of CCR7 on CD4⁺ T cells in PBMCs of the subject is higher than2402, (d) the measured frequency of Ki67⁺EOMES⁺CD4⁺ T cells in PBMCs ofthe subject is less than 0.446%, (e) the measured expression level ofTGFβR3 on CD8⁺ T cells in PBMCs of the subject is higher than 527, (f)the expression level of CD109 on CD8⁺ T cells is greater than 0.7315%,(g) the frequency of CD71⁺CD4⁺ T cells in CD4⁺ T cells in the PBMCs isless than 2.80%, or a combination.
 43. A method of treating a subjectdiagnosed with melanoma with both a CTLA-4 blockade treatment and adifferent treatment if (a) the measured frequency of Ki67⁺EOMES⁺CD8⁺ Tcells in peripheral blood mononuclear cells (PBMCs) of the subject isless than 2.11%, (b) the measured frequency of EOMES⁺CD8⁺ T cells inPBMCs of the subject is less than 55.6%, (c) the measured expressionlevel of CCR7 on CD4⁺ T cells in PBMCs of the subject is higher than2402, (d) the measured frequency of Ki67⁺EOMES⁺CD4⁺ T cells in PBMCs ofthe subject is less than 0.446%, (e) the measured expression level ofTGFβR3 on CD8⁺ T cells in PBMCs of the subject is higher than 527, (f)the expression level of CD109 on CD8⁺ T cells is greater than 0.7315%,(g) the frequency of CD71⁺CD4⁺ T cells in CD4⁺ T cells in the PBMCs isless than 2.80%, or a combination.
 44. The method of any one of claims32-43, wherein the CTLA-4 blockade treatment is treatment withipilimumab or tremelimumab.
 45. The method of any one of claims 32-43,wherein the CTLA-4 blockade treatment is treatment with ipilimumab.